Down-regulation of the let-7i facilitates gastric cancer invasion and metastasis by targeting COL1A1
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Globally, gastric cancer is the most common malignant tumor and the second highest contributor to cancer deaths after lung cancer (Murray et al., 2012). Despite improved success with treatment of early stage gastric cancer (Fuse et al., 2016), the five-year survival rate of advanced staged gastric cancer patients is still low. The aggressive growth characteristics of the tumor and metastasis are key factors responsible for poor overall survival in these patients (Ozkan et al., 2005). Therefore, investigation of the molecular mechanisms that underlie the aggressive behavior of gastric cancers, and identification of potential target genes for therapeutic interventions, is a key imperative.
Aberrant miRNA expression is a key contributor to tumorigenesis in humans (Croce and Calin, 2005; Iorio et al., 2005; Wu et al., 2009; Acunzo and Croce, 2016). Our previous study showed that as a crucial hub gene in gastric cancer, COL1A1 is directly regulated by let-7i miRNA and its high expression levels in gastric cancer have been linked to increased tumor invasiveness (Shi et al., 2015). Downregulation of Let-7i in several cancers was shown to be associated with unfavorable prognosis (Yang et al., 2008; Yang et al., 2013). However, whether let-7i influences progression of gastric cancers is not known.
In the present study, we assessed the let-7i expression level and its effects in gastric cancer samples and cell lines. The binding sites of COL1A1 and let-7i were predicted using bioinformatics software and their regulatory mechanism verified. Further, we also analyzed expression levels of COL1A1 in gastric cancer tissues and cell lines. The results suggest that increased let-7i expression may lead to decrease in proliferative, metastatic and invasive properties of cancer cells.
In our previous microarray study, up-regulation of COL1A1 in gastric cancers was significantly associated with invasiveness of gastric cancer (Shi et al., 2015). By applying the TF-miRNA co-regulatory network, we established COL1A1 to be a target gene of let-7i. Thus, we inferred that the expression levels and functional mechanism of COL1A1 might be associated with the regulatory effects of let-7i. Being a recently discovered member of the let-7 family, functional characterization of let-7i is not well-investigated. A recent study showed an association of low expression of let-7i in gastric cancer with tumor cell invasion and lymph node metastasis (Liu et al., 2012). Yang et al. reported an increased susceptibility of ovarian cancer cells to chemotherapy after up-regulation of let-7i (Yang et al., 2008). Blower et al., too, reported increased or decreased sensitivity of tumor cells to chemotherapy drugs after up-regulation or down-regulation of let-7i expression in NCI-60 cancer cell lines, respectively (Blower et al., 2008). In the current study, we observed significant down-regulation of let-7i expression in gastric cancer tissue specimens as compared to that in the corresponding normal tissue specimens. Since, down-regulated let-7i expression was significantly associated with lymph node metastasis and advanced T stage, these results suggest that let-7i may have a suppressive role in progression of gastric cancer. Let-7i was also significantly down-regulated in gastric cancer cell lines (SGC-7901, MGC-803, AGS, N87) as compared to that in normal gastric epithelial cell line GES-1. Let-7i restoration reduced gastric cancer cell viability, migration and invasive properties in vitro. Furthermore, restoration of let-7i expression inhibited growth of nude mouse xenografts in vivo.
Collagen is an important component of ECM that occurs as collagen types I, II and III in the highest proportions. As a member of the collagen I family, association of COL1A1 with tumor cell proliferation and invasion has been reported in many cancers such as breast, lung and renal cancers (Boguslawska et al., 2015; Grigoroiu et al., 2015; Chai et al., 2016). In our previous study, we demonstrated that up-regulated expression of COL1A1was closely associated with invasive properties of gastric cancer cells. In this study, we found that COL1A1 expression was inversely associated with let-7i levels in gastric cancer tissues. Level of COL1A1 mRNA and protein expression was significantly reduced after ectopic expression of let-7i in gastric cancer cells. Luciferase activity assay further demonstrated that COL1A1 was a direct target of let-7i. Moreover, knockdown of COL1A1 in gastric cancer cells curbed the proliferative, migratory and invasive ability of cancer cells, which was similar to the results obtained with artificial upregulation of let-7i. Moreover, the restoration of COL1A1 expression in cells stably expressing let-7i was able to counteract the inhibitory effects of let-7i in gastric cancer cells. Taken together, our findings provide strong evidence of COL1A1 as being a direct and functional target of let-7i.
In summary, the present study provides strong evidence of suppressive effects of let-7i on gastric cancer development and progression. Ectopic expression of let-7i successfully suppressed multiple malignant biological characteristics including inhibition of tumor cell viability and reduction of migration and invasion in vitro and tumor xenograft growth in vivo. Our further experiments revealed that overexpressed COL1A1 was a direct target of let-7i in gastric cancer cells. Knockdown of COL1A1 in gastric cancer cells produced an anti-proliferative, migratory and invasive reduced effect, which was similar to the results when let-7i up-regulation. These results suggest a potential application of let-7i as a diagnostic and therapeutic target for gastric cancers. Future studies will seek to confirm the functional mechanisms of let-7i in gastric cancer.
This work was supported by grants from National Natural Science Foundation of China (Grant Nos. 81320108025, 81472662 and 81672109), foundation of Jilin Province Science and Technology Department (172408GH010234983), and Jilin University-Xinjiang Medical University joint research project.
Yue Shi, Zipeng Duan, Xun Zhang, Xiaotian Zhang, Guoqing Wang and Fan Li declare that they have no conflict of interest.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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