Let-7g targets collagen type I alpha2 and inhibits cell migration in hepatocellular carcinoma - PubMed (original) (raw)

Let-7g targets collagen type I alpha2 and inhibits cell migration in hepatocellular carcinoma

Junfang Ji et al. J Hepatol. 2010 May.

Abstract

Background & aims: Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis mainly due to metastasis. MicroRNAs are endogenous small noncoding RNAs that regulate cellular gene expression and are functionally linked to tumourigenesis. Using microarray analysis, we recently identified 20 miRNAs associated with HCC metastasis. Here, we carried out further analyses on one of these microRNAs, let-7g, to determine whether it is functionally linked to HCC metastasis.

Methods: Quantitative real-time polymerase chain reaction was used to determine the level of mature let-7g transcript in HCC clinical specimens and its correlation with patient survival. Ectopic expression of let-7g was carried out in HCC cell lines to assess its influence on cell growth, migration, and invasion.

Results: We confirmed that the level of let-7g was significantly lower in metastatic HCCs compared to metastasis-free HCCs. Moreover, low let-7g expression in a tumour was predictive of poor survival in HCC patients. Functional studies indicated that ectopic expression of let-7g significantly inhibits HCC cell migration and cell growth. In-silico analysis revealed members of soluble collagens as potential targets of let-7g. Consistently, the levels of type I collagen alpha2 (COL1A2) and let-7g were inversely correlated in HCC clinical specimens. COL1A2 was experimentally validated as a direct target of let-7g. Moreover, addition of COL1A2 counteracted the inhibitory effect of let-7g on cell migration.

Conclusions: These results suggest that let-7g may suppress HCC metastasis partially through targeting COL1A2.

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Figures

Figure 1. Let-7g was uniquely down-regulated in HCC cases with metastasis and was predictive of survival

(A) The relative expression levels of let-7g from metastatic HCC cases (n = 11) and non-metastastic HCC cases (n = 11) were determined by qRT-PCR. Un-paired _t_-tests were used. (B) A linear regression and correlation among data from qRT-PCR versus microarray is shown with r (spearman) and p-values indicated (right panel). Expression status is shown as the tumour/non-tumour ratio in a log2 scale. (C) The association of tumour vs. non-tumour let-7g expression with overall survival of 22 HCC patients (high let-7g (tumour/non-tumour ≥ 1), n = 10; low let-7g (tumour/nontumour <1), n = 12). (D) The association of tumour vs. non-tumour let-7g expression with overall survival of 33 HCC patients (high let-7g, n = 12; low let-7g, n = 21).

Figure 2. Inhibition of cell migration in huH1 cells by let-7g in an in vitro cell migration assay

(A) The expression levels of let-7g in huH-1, huH1-miRCtrl and huH1-let7g cells determined by qRT-PCR. (B) huH1-miRCtrl and huH1-let7g cells were subjected to a cell migration assay. Representative images of migrated cells are shown in the left panel and quantitative data are shown in the right panel. (C) A cell invasion assay was performed on huH1-miRCtrl and huH1-let7g cells. Representative images of migrated cells are shown in the left panel and quantitative data are shown in the right panel. Data from A-C are shown as the mean ± SD derived from three independent experiments, and Student’s _t_-test was used.

Figure 3. Inhibition of cell migration in huH1 cells by let-7g in an in vitro wound healing assay

Scratches were generated in a confluent monolayer of huH1-miRCtrl or huH1-let7g cells and the degree of “wound remaining” was measured after 1 hr, 8 hr, 24 hr and 32 hr. (A) Representative images of the wound gaps in controls and huH1-let7g cells at each time point. (B) Percent wound remaining is shown as the mean ± SD of three experiments.

Figure 4. Inhibition of HCC cell growth by let-7g

(A) A colony formation assay in huH1-miRCtrl and huH1-let7g cells was performed, and representative dishes of huH1-miRCtrl and huH1-let7g cells are shown in the left panel and quantitative data are in the right panel. (B) A cell proliferation assay in huH1-miRCtrl and huH1-let7g cells. The percentage of cells forming colonies (A) and the relative cell number (B) are shown as the mean ± SD of three experiments.

Figure 5. Direct targeting of COL1A2 by let-7g

(A) Collagen assays were performed in huH1-miRCtrl and huH1-let7g cells. Relative expression of collagens type I to V is shown as the mean ± SD. (B) Predicted duplex formation between the 3’-UTR sequences of human COL1A2 and let-7g. The underlined bases of let-7g highlight the seed sequences of this miRNA. The underlined bases in the COL1A2 3’-UTR were targeted for mutation analysis when inserted into a luciferase reporter. (C) The expression of COL1A2 at the mRNA (right) and protein (left) levels in huH1-miRCtrl and huH1-let7g cells. (D) Luciferase activity of various reporter plasmids in huH1-miRCtrl and huH1-let7g cells. The luciferase activity is shown as mean ± SD. (E) A wound healing assay was performed in huH1-miRCtrl and huH1-let-7g cells with and without the presence of 0.2 µg/ml of type I collagen. The wound healing assay was done in 20 hours. Statistical significance was calculated using Student’s _t_-test. * refers to p <0.05.

Figure 6. COL1A2 expression was negatively correlated with let-7g level in HCC cases

(A) The relative expression levels of COL1A2 from metastatic HCC cases (n = 11) and non-metastatic HCC cases (n = 11) from microarray data. (B) A linear regression and correlation among let-7g versus COL1A2 data is shown with r (spearman) and p-value indicated (right panel). Expression status is shown as the tumour/non-tumour ratio in a log2 scale.

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Let-7g targets collagen type I alpha2 and inhibits cell migration in hepatocellular carcinoma - PubMed (original) (raw)