miRNAs are intricated in several biological processes and play a noteworthy role in pathological approaches. In several types of cancers, miRNAs are mainly responsible for tumorigenesis, cancer progression, cell invasion and metastasis [14]. When compared with colon cancer, the existing studies pertinent to rectal cancer are relatively less. While there are some previous approaches to identify the role of miR-652 in cirrhosis of liver, focusing on their control in liver tissue has not been characterized in humans or rodents [15]. In the present study, we examined whether selected candidate miRNAs could serve as a marker for rectal cancer by analyzing the relative level of miR-652 in samples from 101 rectal cancer patients who participated in a Swedish trial of preoperative RT.
In plasma of gastric cancer patients, a miRNA-microarray platform was used to screen the differentially expressed miRNAs and upregulated miR-652 was further selected for validation [16]. However, expression levels of miR-652 were found to be downregulated in primary squamous cell lung carcinoma [17].
In the present study, miR-652 expression was increased significantly in primary cancer than normal mucosa in non-RT patients. Further, high miR-652 expression in non-RT cases was positively associated with apoptosis, ATM expression and DNp73 expression. A previous study exhibited that ATM expression level was significantly increased in colorectal cancer [18]. Likewise, the increased expression of DNp73 has been observed in several types of tumors and cell lines, and it is linked to pro-tumor activities [19]. Furthermore, we found that the high level of miR-652 expression was independently related to worse DFS non-RT patients.
To validate the role of miR-652 expression, we used the TCGA database and identified a high significant expression in rectal cancer tissue. Prognostic analysis showed that expression of miR-652 was independently related to unfavorable survival of patients. The results from the TCGA database were in accordance with those in our samples. To further determine the biological function of miR-652, MiRNet was used for network creation, and analysis and a lot of related genes were shown. GO annotation showed that miRNA catabolic process and RNA catabolic process were found to be highly related to miR-652 regulated genes. The ribosome pathway was the only significant related pathway for miR-652 related genes from KEGG pathway analysis. In the miRNA-gene network, several genes, including LLGL1, INO80D, MTFP1, and ZFAND5 were significantly related to miR-652. A previous study showed that downregulation of LLGL1 was associated with the progression of colorectal cancer (CRC) [20]. INO80D is a subunit of the human INO80 chromatin-remodeling complex and is intricated in the regulation of transcription, DNA replication as well as repair mechanism [21]. For MTFP1, the loss of function instigates the cytochrome c release, which activates caspase cascade and further leads to apoptosis [22–23]. ZFAND5 plays an important role in controlling NF-kappa-B activation and apoptosis [24].
In RT patients, miR-652 expression was significantly decreased in cancers when compared to non-RT cases. However, the level of miR-652 expression was negatively related to WRAP53 expression. Previous studies showed that COX-2 was instigated by p53-mediated activation of RAS or RAF or ERK cascade [25], and WRAP53 encoded a regulatory RNA required for the function of p53 upon DNA damage [26].
To further explore the underlying mechanism by which miR-652 response to radiation, the ROS estimation, caspase activity, and apoptosis were determined by flow cytometry after radiation and miR-652 inhibition. It showed that the ROS estimation, caspase activity and apoptosis in HCT116 p53+/+ cells rather than HCT116 p53-/- cells were significantly increased after radiation. Further studies are needed to determine whether miR-652 was involved in p53-dependent or -independent apoptosis induced by radiation.