MicroRNA-224 Promotes Cell Migration and Invasion by Targeting HOXA5 expression in Hepatocellular Carcinoma

MicroRNAs (miRNAs) are involved in the regulation of multiple cellular pathways and play a key role in the development and progression of tumor. Based on the cellular function of their targets, miRNAs play the role of oncogenes or tumor suppressor genes. Multiple studies have shown that abnormal expression of miRNAs has close relation with the incidence of HCC, but the mechanism of miRNAs in HCC still needs further research. In the present study, we showed that the overexpression of miR-224 can reduce the mRNA and protein expression of ADAM17 and HOXA5, the silencing of miR-224 can increase the protein expression of ADAM17 and HOXA5. Dual luciferase reporter assays showed that miR-224 can directly regulate the expression of ADAM17 and HOXA5. Importantly, we found that miR-224 positively regulates cell migration and invasion in HCC, miR-224 overexpression can promote the migration and invasion of BEL-7402 cell, and miR-224 silencing can suppress the migration and invasion of BEL-7402 cell. miR-224 overexpression can result in the redistribution of cell cycle, the cell percentage of S phase was increased significantly, the cell percentage of G1 phase was decreased significantly, and there is no noticeable change for the cell percentage of G2 phase. These results demonstrated that it may be exert the function of oncogenes in a particular link of cancer cell growth. In conclusion, these results suggest that miR-224 will become a promising biological target in the treatment strategy of hepatocellular carcinoma (HCC).

precursors. Among distantly related organisms, the sequence of miRNAs is 5 conserved, suggesting that these molecules are involved in the basic biological 6 processes of the organism [1][2][3]. To date, more than 500 miRNAs have been 7 predicted to be expressed in humans [4,5], and these miRNAs are estimated to 8 regulate the expression of over 5,000 human genes or 30% of human proteins [6]. 9 Interactions between MiRNAs and their numerous target genes may represent 10 important levels of gene regulation in cells [7]. Although the exact function of miRNAs 11 is still unknown, existing studies have shown that miRNAs have a variety of different 12 expression patterns and can modulate many different developmental and 13 physiological processes. In addition, dysregulation of miRNAs expression may lead to 14 human disease [8]. Several studies have shown that close to 50% of miRNAs genes 15 are located in cancer-related genomic regions or fragile sites, which draws attention to 16 the importance of miRNAs in cancer [9][10][11].

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Abnormal expression of MiRNAs has been shown to involve a variety of solid 18 tumors, including hepatocellular carcinoma, colon cancer, prostate cancer, breast 19 cancer, ovarian cancer and lung cancer [12][13][14][15]. In addition, abnormal expression of 20 miRNAs has been found to be associated with postoperative survival of lung cancer 21 patients [16], and can be used as a biomarker for the diagnosis and treatment of lung 22 cancer [17]. According to the role of their target genes, miRNAs can play an important 4 1 role in the occurrence and development of tumors as both tumor suppressor genes 2 and oncogenes [18]. The use of gene chip make it possible for the analysis of 3 large-scale miRNAs expression in tumor study, we can confirm a lot of miRNAs in 4 cancer tissues and normal tissues in the differences in expression by this way, so that 5 we can better explain the imbalance in the development of tumor miRNAs. Although 6 the function of some cancer-related miRNAs has been revealed, the mechanism of 7 action of most miRNAs in tumors remains unclear [19]. 8 Some miRNAs have been proved to have the function of tumor suppressor genes. 9 Johnson SM et al [20] reported that the let family can regulate the expression of RAS 10 gene, revealing that it may play an inhibitory role in the development of lung cancer. O

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'donnell et al [21] reported that miR-17-5p and miR-20a negatively regulate the 12 expression of E2F1, thereby affecting the function of c-myc gene and ultimately 13 controlling the signals of proliferation. Some miRNAs have been shown to have the 14 function of oncogenes, and miR-21 has been found to be up-regulated in both glioma 15 and breast cancer and to play an anti-apoptotic role [22,23] Although we have some understanding of the expression and function of miR-224 3 in hepatocellular carcinoma based on some previous reports, the exact mechanism or 4 intracellular target of miR-224 in hepatocellular carcinoma is still poorly understood. 5 Therefore, the detection of the target of miR-224 is very important for our in-depth 6 understanding of the mechanism of action of miR-224. In this study, we will further 7 investigate the mechanism of miR-224 in the development of hepatocellular 8 carcinoma. We screened the target genes of miR-224 through bioinformatics, and 9 selected ADAM17 and HOXA5 as the starting points of this study, hoping to find 10 strong evidence that miR-224 plays an important role in the development of 11 hepatocellular carcinoma. The clinicopathologic features of 42 patients with paraffin-embedded hepatocellular 9 carcinoma samples were described briefly (S1 file ). All of 42 patients contained 37 10 men and 5 women, the sex ratio( male/female) was 7:1. The mean age was 50 yeas 11 old. According to microscopic pathological characteristics, histological grade of tumor 12 differentiation was assigned: 17% were well-differentiated, 57% were    ] method with their endogenous control.

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The whole construction process was completed by Shanghai jima pharmaceutical 4 technology co., LTD. The corresponding sequence information is as follows: Experiments were repeated at least three times. The two-tailed student's t-test, one-way ANOVA, Mann-Whitney U or Kruskal-Wallis

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Test were used for data analysis. The statistical analyses were done using SPSS 19.0 20 for windows. P<0.05 was considered significant.

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The expression of miR-224 in hepatocellular carcinoma and adjacent tissues 23 Expression of miR-224 was detected in 19 pairs of human hepatocellular carcinoma 12 1 and adjacent liver tissues by real-time quantitative RT-PCR. Expression of miR-224 2 was upregulated in 2 cases of hepatocellular carcinoma tissues (>1.5 fold); It is 3 considered to be virtually unchanged in 3 cases of hepatocellular carcinoma tissues, 4 because the magnitude of increased or decreased miR-224 expression was less than 5 1.5 fold; the down-regulation of miR-224 was found in 14 cases of hepatocellular 6 carcinoma tissues (>1.5 fold), the statistical analysis showed that the expression of 7 miR-224 in hepatocellular carcinoma tissues was significantly higher than that in 8 adjacent liver tissues (P<0.01, Mann-Whitney U test) (Fig 1A). These results suggest 9 that miR-224 may play a role as an oncogene in hepatocellular carcinoma. In order to better understand the mechanism of miR-224 in the development of HCC, to predict the target genes of miR-224. Among the many predicted target genes, 4 ADAM17 and HOXA5 were selected as the targets for further study. The 3'UTR region 5 of the mRNA encoded by ADAM17 and HOXA5 genes contains sequences that are 6 partially complementary to miR-224 (Fig 2). two-tailed Student's t-test) (Fig 3). in Huh-7 cells (Fig 4).

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In order to detect the direct interaction between miR-224 and its target genes 5 ADAM17 and HOXA5, we co-transfected miR-224 mimics or mimics NC with wild-type 6 or mutant vectors of ADAM17 and HOXA5 into 293T cells, and then detected the 7 changes of double luciferase of the vector. We found that the fluorescence activity of 8 the wild-type reporting genomes of ADAM17 and HOXA5 was significantly lower than 9 that of the control group (P<0.05, one-way ANOVA) (Fig 5). This result suggests that 10 miR-224 can directly regulate ADAM17 and HOXA5.

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Combining the effects of miR-224 mimics and inhibitor on three strains of HCC, compared with the inhibitor NC group, the ability of miR-224 inhibitor group to migrate 4 (P <0.01, the two-tailed Student's t-test) (Fig 6B) and to invade (P <0.01, the 5 two-tailed Student's t-test) (Fig 7B) was significantly reduced. Student's t-test) (Fig 8). t-test (Fig 9A).In addition, after miR-224 was silenced, flow results showed that the 2 cell percentage of miR-224 inhibitor group in G1, S and G2 phases did not change 3 significantly compared with that of inhibitor NC group (P>0.05, all, the two-tailed 4 Student's t-test) (Fig 9B).These results suggest that miR-224 overexpression can 5 promote the proliferation of bel-7402 cells. . These results suggest that the mechanism of miR-224 in tumors is 9 complex, requiring further study. 10 In this study, we used a variety of methods to identify and confirm the target genes of should also be fully considered.

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Using bioinformatics to predict miRNAs target genes is a convenient method for 10 preliminary screening of target genes, which can provide us with many candidate 11 target genes. Among these predicted target genes, many target genes have been