Targeting FHL2-E-cadherin axis by miR-340-5p attenuates colon cancer cell migration and invasion

Abstract

Convincing data has suggested that four and a half LIM domain 2 protein (FHL2) serves a key function in cancer cell metastasis and that microRNA (miR)-340-5p can regulate cancer cell migration. The current study hypothesized that targeting FHL2 expression by miR-340-5p in colon cancer may attenuate colon cancer cell migration and invasion. FHL2 expression was therefore assessed in colon cancer microarray datasets using Qlucore omics explorer as well as in HT-29 and AZ-97 colon cancer cell lines via reverse transcription-quantitative PCR (RT-qPCR). Colon cancer cell migration and invasion were evaluated in the presence of miR-340-5p mimic, mimic control or mimic with a target site blocker. Confocal microscopy and RT-qPCR were subsequently performed to assess FHL2, E-cadherin (E-cad) protein and mRNA expression in colon cancer cells. Microarray dataset analysis revealed that FHL2 expression was lower in primary colon cancer cells compared with normal colonic mucosa. It was revealed that the expression of miR-340-5p and FHL2 were inversely related in serum-grown and low-serum conditions in HT-29 and AZ-97 cells. Short-time serum exposure to low-serum grown cells induced FHL2 expression. Transfection of HT-29 cells with miR-340-5p mimic not only decreased serum-induced expression of FHL2 but also decreased cancer cell migration and invasion. Bioinformatics analysis revealed that FHL2 mRNA had one putative binding site for miR-340-5p at the 3-untranslated region. Blocking of the target site using a specific blocker reverted miR-340-5p mimic-induced inhibition of FHL2 expression and cancer cell migration and invasion. Confocal microscopy confirmed that the reduction of FHL2 expression by miR-340-5p mimic also reversed serum-induced E-cad disruption and that the target site blocker abrogated the effect of miR-340-5p. The current results suggested that miR-340-5p could be used to antagonize colon cancer cell metastasis by targeting the FHL2-E-cad axis.

Keywords: cell migration; colon cancer; four and a half LIM domains protein 2; metastasis; microRNAs.

Figure 1.

Expression of FHL2 in colon cancer from different microarray datasets. FHL2 expression was compared in normal colon mucosa and colon cancer tissue in (A) GDS4382 and (B) GSE115313 datasets. FHL2 expression was also compared in patients with primary colon cancer, metastatic colon cancer and recurrence metastasis in (C) GDS4393 and (D) GDS4516 datasets. Box plots were created using Qlucore program and represent the mean (25–75 percentile), with whiskers indicating the minimum and maximum levels, and dots represent log₂ normalized sample expression values. FHL2, four and a half LIM domains protein 2

Figure 2.

Expression of miR-340-5p and FHL2 in HT-29 and AZ-97 colon cancer cell lines. Expression of (A) miR-340-5p and (B) FHL2 mRNA in low-serum and serum-grown HT-29 and AZ-97 cells. Relative expressions were determined using reverse transcription-quantitative PCR with U6 as a control for miR-340-5p and β-actin as a control for FHL2 mRNA. Relative expressions were determined using the 2^−ΔΔCq method. Data are presented as the mean ± SEM (n=4). miR, microRNA; FHL2, four and a half LIM domains protein 2.

Figure 3.

miR-340-5p regulates FHL2 mRNA expression in HT-29 and AZ-97 colon cancer cells. Transfection with 50 nM miR-340-5p mimic (A) upregulated miR-340-5p and (B) downregulated FHL2 mRNA expression in colon cancer cells. Relative expressions were determined by performing reverse transcription-quantitative-PCR where U6 was used as a a control for miR-340-5p and β-actin was used as a control for FHL2 mRNA. Relative expressions were determined using the 2^−ΔΔCq method. Data are presented as the mean ± SEM (n=4). miR, microRNA; FHL2, four and a half LIM domains protein 2; Ctrl, control.

Figure 4.

miR-340-5p directly targets FHL2. (A) The predicted target site of miR-340-5p in the 3′-UTR sequence of FHL2 mRNA containing an AAUAUUUC motif is presented. The seeding region of miR-340-5p complementary to UUAUAAAG was then blocked using TSB (red sequence). (B) TSB (50 nM) reversed the inhibitory effect of miR-340-5p (50 nM) on FHL2 mRNA expression in HT-29 colon cancer cells. Data are presented as the mean ± SEM (n=4). miR, microRNA; FHL2, four and a half LIM domains protein 2; TSB, target site blocker; nt, nucleotide; Ctrl, control.

Figure 5.

miR-340-5p downregulates FHL2 expression in colon cancer cells. FHL2 expression was evaluated using (A and B) RT-qPCR and (C-F) confocal microscopy (scale bars, 10 µm). Cells were transfected with miR-340-5p mimic, mimic control, TSB control and TSB for 24 h in low-serum conditions. Colon cancer cells were then stimulated using 10% FBS for 30 min. Relative expressions of FHL2 mRNA was quantified using RT-qPCR where β-actin was used as an internal control for FHL2 mRNA. Expressions were determined using 2^−ΔΔCq method. Data are presented as the mean ± SEM (n=4). miR, microRNA; FHL2, four and a half LIM domains protein 2; RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; Ctrl, control.

Figure 6.

miR-340-5p regulates colon cancer cell migration and invasion. Cells were transfected with miR-340-5p mimic, mimic control, TSB control and TSB for 24 h in low-serum conditions. Serum-stressed transfected cells were then loaded in the upper chamber of the trans-well equipment, with the lower chamber filled with 10% bovine serum albumin. (A) Migration of colon cancer cells toward serum. (B) Invasion of colon cancer cells towards serum. Cells were counted microscopically in five different fields. Data are presented as the mean ± SEM (n=4). miR, microRNA; TSB, target site blocker; Ctrl, control; HPF, high power field.

Figure 7.

miR-340-5p upregulates E-cad expression in colon cancer cells. E-cad expression was evaluated using (A and B) RT-qPCR and (C-F) confocal microscopy (scale bars, 10 µm). Cells were transfected with miR-340-5p mimic, mimic control, TSB control and TSB for 24 h in low-serum conditions. Colon cancer cells were then stimulated using 10% FBS for 30 min. Relative expressions of E-cad mRNA were quantified using RT-qPCR where β-actin was used as an internal control. Expressions were determined using 2^−ΔΔCq method. Data are presented as the mean ± SEM (n=4). miR, microRNA; E-cad, E-cadherin; RT-qPCR, reverse transcription-quantitative PCR; TSB, target site blocker.