Introduction
Cervical cancer is the second most common cancer in women worldwide. Approximately 80% of primary cervical cancers arise from pre-existing squamous dysplasia. The most important etiologic agent in the pathogenesis is human papillomavirus (HPV). However, not all women infected with high-risk HPV develop cervical carcinoma. It is obviously that many genetic and epigenetic alternations occur during its tumorigenesis. Among those changes, aberrant promoter methylation of tumor-suppressor genes gives rise to its silencing functions and results in the significant carcinogenesis of cervical cancer.
Until recently, few evidences have shown that miRNA genes may be regulated by epigenetic mechanisms, as changes in genomic DNA methylation pattern. In this study, we present the results of a genome-wide miRNA expression in cervical cancer cells treated with 5-aza-2’-deoxycytidine and identify the altered methylation of miRNA genes as a possible epigenetic mechanism responsible for their aberrant expression.
Discussion
MicroRNA (miRNA) is a novel class of short non-coding RNA molecules regulating a wide range of cellular functions through translational repression of their target genes. DNA methylation is one of the heritable epigenetic modifications, repressing gene expressions and consequent phenotypic alterations without changing the DNA sequence. Recently, epigenetic dysregulation of tumor suppressor miRNA genes by promoter DNA methylation has been implicated in human cancers.
In our previous study, we found miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. We also proved that miR-21 is involved in cervical squamous cell tumorigenesis by CCL20 (Yao T, et al., [
10]). 5-Aza-CdR which can reactivate the expression of methylated tumor suppressor genes, is a pyrimidine nucleoside analog that forms a covalent complex with DNMT during DNA replication (Yao TT, et al., [
11]). To evaluate if an aberrant DNA methylation pattern could also contribute to the altered miRNA expression characterizing the cervical carcinoma, we analyzed the miRNA profiling of the cervical cancer cell lines before and after treatment with 5-AZA. Resulting from our analyses, a number of miRNAs were over-expressed but not reported, as well as down-modulated but not deleted. The involvement of an epigenetic regulatory mechanism could actually exert a role on miRNA expression in human cervical cancer.
It is conceivable that several disregulated miRNAs with no defined functions at this point may exhibit tumor suppressor or oncogene activity and normally target components of key signaling pathways that promote and maintain the growth and survival of cells. Several analysis revealed high expression level of miR-432 which remained significant as independent predictor for recurrence-free (RFS) of hepatocellular carcinoma (Huang YH, et al., [
12]). However, in human pituitary GH adenomas, miR-432 plays a role as tumor suppressor gene by regulating HMGA2 (D’Angelo D, et al., [
13]).
Thirteen miRNAs including of miR-1286 were deregulated in both two esophageal carcinoma cell lines (one adenocarcinoma and one squamous cell carcinoma) treated with cisplatin or 5-fluorouracil for 24 or 72 h (Hummel R, et al., [
14]). Analysis of predicted targets of it highlighted molecular networks included functions such as ‘Cell death’, ‘Cell cycle’, ‘Cellular growth and proliferation’, ‘DNA replication, recombination, and repair’ and ‘Drug metabolism’. Cisplatin or 5-fluorouracil altered miRNA expression in esophageal cancer cells. Ingenuity Pathway Analysis (IPA) suggested that miR-1286 may target molecular pathways involved in cell survival after chemotherapy.
Until recently, there has been just one report about miR-641 up-regulated in normal chondrocytes (Díaz-Prado S, et al., [
15]).
In silico, analyses predicted that key molecular pathways potentially altered by miRNAs differentially expressed in normal and OA chondrocytes include TGF-beta, Wnt, Erb and mTOR signalling; all of them implicated in the development, maintenance and destruction of articular cartilage. We concluded the up-modulation of miR-641 in our study may also have relationship with these signaling.
Researches on miR-1290 have been referred on tumor therapy. Following in vitro photodynamic treatment, miR-1290 was overexpressed in human epidermoid carcinoma cells (A431) (Bach D, et al., [
16]). Up-regulation of microRNA-1290 impairs cytokinesis and affects the reprogramming of colon cancer cells (Wu J, et al., [
17]). In light of these reports and our results, we proposed miR-1290-based therapeutic approaches could be developed in future.
Wilcoxon sign-rank test for paired samples analysis revealed that abnormal miRNAs showed a higher level of variation across different breast cancer samples. Most of these miRNAs were significantly down-regulated in tumor samples, but miR-1287 was consistently expressed in tumor tissues and serum samples (Guo L, et al., [
18]). In follicular lymphoma, it was increased (Wang W, et al., [
19]).
MiR-95 has been shown to be involved in carcinogenesis. A highly characterized example is pancreatic cancer, in which miR-95 was confirmed significantly increasing cell proliferation, invasion, migration and inhibited cell apoptosis in vitro and in vivo when compared with negative control (Li WG, et al., [
20]). Concordant changes were also revealed in colorectal carcinoma (Huang Z, et al., [
21]) and pancreatic cancer (Zhang Y, et al., [
22]). However in head and neck cancers, miR-95 has been suggested downregulated (Nurul-Syakima AM, et al., [
23]). In HeLa cells, inhibition of miR-95 caused a decrease in cell growth (Cheng AM, et al., [
24]).
In our study, we found miR-625 was decreased after demethylation. Increasing evidence points to a central role of miR-625 for vesicle trafficking processes in oncogenesis and tumor suppression. Expression of miR-625 is significantly down-regulated and negatively correlated with lymph node metastasis in gastric cancer. MiR-625 significantly inhibits invasion and metastasis of gastric cancer cells both in vitro and in vivo. ILK is a direct target gene for miR-625 and knockdown of ILK has a phenocopy of overexpression of miR-625 (Wang M, et al., [
25]). In the hormonal treatment of endometrial carcinoma, Hec1A cells were treated with medroxyprogesterone acetate, the expression levels of miR-625 was increased by more than 400% (Bae J, et al., [
26]).
The methlyation DNA immunoprecipitation-based chip analysis (MeDIP-chip) is a novel high- throughput array-based method using a monoclonal antibody against 5-methycytidine for the enrichment of the methylated DNA fragments and then hybridizing to the promoter and CpG islands of the entire human genome (Zhang Z, et al., [
27]). Our study found that the contents of promoter methylation in miR-641 and miR-1287 were inversely correlated with the expression levels of these genes in cells treated with 5-aza-2’-deoxycytidine. Contradictorily, our results showed that after treatment of 5-AZA, miR-95 was significantly up-regulated in cell lines, whereas the expression of it was lower in cervical cancer than normal tissues. We proposed there may be some other factors coexisted.
However, these different human miRNAs were not found in microarray of C33A. So, we concluded that the downregulation may have relationship with the negative expression of HPV in C33A. It is intriguing to speculate that the expression of cellular miRNA genes at or near HPV integration sites may contribute to the tumor phenotype.The involvement of an epigenetic regulatory mechanism could actually exert a role on miRNA expression in cervical cancer. Apparently, our findings may provide new insights into understanding the pathogenesis of cervical cancer.
Competing interests
The authors declare no conflict of interest.
Authors’ contributions
ZL was responsible for study design. MY was responsible for the experiment, data analysis, literature search and article drafting. Other authors were responsible for histopathological information collection of all data. All authors read and approved the final manuscript.