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.
Highlights
Colon cancer is the second most frequent cause of cancer‐related death in the world [1]
We analyzed FHL2 expression among some common colon cancer cell lines and found that FHL2 expression was relatively low in HT‐29 and HCC2998 cell lines compared to other cell lines such as CaCo2, DLD‐1, HCT116, HCT15, LoVo, SW480 and TC71 (Fig. S1A)
Knowing that FHL2 levels are different in different cancer types, we analyzed a dataset (GSE103512) containing four different cancer samples, our analysis revealed that colon cancer and prostate cancer had similar levels of FHL2 expression while breast cancer and non‐small cell lung cancer had significantly lower levels of FHL2 expression than colon cancer and prostate cancer (Fig. S2)
Summary
Colon cancer (colon and rectal) is the second most frequent cause of cancer‐related death in the world [1]. The overall prog‐ nosis of colon cancer has improved over the past decades owing better surgical techniques as well as improved neo‐adjuvant and adjuvant treatment. 20% of colon cancer patients have metastatic disease at diag‐ nosis and more than 30% of patients with colon cancer develop metastasis over time [3]. Several studies have shown that stress conditions such as hypoxia, lack of nutrients and pH altera‐ tions can induce up‐regulation of pro‐metastatic genes [4,5,6]. Alterations of these genes can subsequently induce cancer cell migration and invasion, further aggravating the disease
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