Background
Hepatocellular carcinoma (HCC) is an extremely common malignant tumor. China is developing one of the highest incidences of liver cancer worldwide. About 45% of de novo HCC cases were discovered in mainland China, and about 110, 000 people died of hepatoma. In fact, in Asian countries such as China, India, the Republic of Korea, Singapore and Vietnam, more than 80% to 90% cases of HCC were associated with human hepatitis B virus (HBV) infections. More than 2 billion people have been infected with HBV worldwide, and more than 350 million of these people became infectors; 75% of all infectors live in Asia, and 33% live in mainland China [
1,
2]. In fact, chronic HBV infection greatly increases the risk of liver cirrhosis and HCC, resulting in the deaths of nearly one million HBV infectors from a variety of liver diseases, such as hepatic failure, hepatic cirrhosis and HCC.
For the past several years, even though the therapy provided to HCC patients has greatly improved, most patients in the middle and advanced stages of HCC have generated portal vein metastases, which form portal vein tumor thrombi (PVTT)[
3]. The resected sample ratio of clinical surgery for the liver is relatively low, and the recurrence ratio after surgery is relatively high [
4]. Overall, the total curative effect in these cases is not as good as expected. The most common reason for carcinoma metastasis is that the cancer cells grow toward the portal vein, which leads to the formation of PVTT. Studies on the mechanisms of tumor formation and metastasis are hindered by difficulties with the corresponding HCC cell lines [
5].
The CXCR4 protein is a G protein-coupled, seven-transmembrane receptor. The chemokine CXCL12, also called stromal-derived factor (SDF-1), is the sole ligand for CXCR4 [
6]. Unlike other chemokines and their receptors, CXCR4 and SDF-1 are constitutively expressed in a variety of tissues, including the brain, heart, liver, lung, spleen and kidney [
1,
7,
8]. SDF-1 is expressed in hematopoietic and non-hematopoietic tissues and was originally identified from bone marrow stromal cells as a pre-B cell growth factor, which is essential for heart, nervous system and blood vessel development. Mice with a targeted deletion of the CXCL12 gene die perinatally, whereas the CXCR4 protein is expressed mainly in neutrophilic granulocytes, macrophages and dendritic cells. The interaction between CXCR4 and SDF-1 plays an important role in the formation of embryos, the development of blood vessels and the heart, the homing of hematopoietic stem cells after transplant, the transmembrane migration of inflammatory cells, T lymphocyte proliferation and the inflammatory response. After further research on the receptor, investigators found that CXCR4 is one of the most comprehensive cytokine receptors expressed in tissue, playing an important role in the growth and metastasis of a variety of malignant tumors [
9].
In this article, through
in vitro primary culture methods, we obtained an HCC cell line derived from the human hepatoma portal vein, which provided the experimental materials for a functional study of the role of CXCR4 in tumor cell invasiveness. To confirm the novel role of CXCR4 in hepatocarcinogenesis, the expression levels of CXCR4 in tumor tissue, adjacent hepatic tissue and PVTT tissue were measured. Finally, the mutual effects of CXCR4 expression and clinical pathology characteristics were discussed [
10]. To further investigate the role of CXCR4 in HCC tumorigenesis and metastasis, a migration assay was performed on PVTT cells following the suppression of CXCR4 expression by the lentivirus-mediated expression of small hairpin RNA (shRNA).
Discussion
In this report, we investigated the differential expression of CXCR4 between PVTT and HCC cells. The expression of CXCR4 in tumor thrombus tissue was higher than in HCC tissue, which was consistent with high expression of CXCR4 facilitating the characteristics of metastasis [
15,
16]. The expression of CXCR4 in HCC tissue was significantly lower than in carcinoma inflammatory liver tissue. As in the data of the group with active hepatitis, the numbers of HBV DNA were all greater than 10
4. Whether or not the adjacent liver tissue was infected with PVTT, in inflammatory conditions CXCR4 was highly expressed.
In the past several years, the establishment of a series of human HCC cell lines provided an ideal
in vitro model to study the pathogenesis of liver cancer, metastasis, development and therapy methods in molecular biology. To explore the role of CXCR4 in carcinoma tissue, the primary cells driven from PVTT have been identified. The tumor cells were mostly derived from the primary HCC tissues of patients. Few studies have used PVTT for establishing cell lines; Hu et al. [
15] reported that the depletion of 8 bp in a chromosome possibly corresponded with the formation of PVTT when using primary cell culture methods on a PVTT that was primarily focused in the liver, as determined by karyotype analysis and comparative genomic hybridization techniques. Our results confirmed one new HCC cell line derived from human PVTT, which provided sufficient experimental support for the study of the formation mechanism of PVTT. In fact, there were nearly no similar references on the establishment of PVTT cell lines for human hepatoma cancer. Therefore, it is important to study the formation and metastasis mechanisms of PVTT in this primary cell line.
To gain insight into the role of CXCR4 in HCC tumorigenesis and metastasis, we employed lentivirus-mediated shRNA to knock down CXCR4 expression in PVTT cells. After screening the siRNA targets, we found the most significant knockdown targeting the expression of CXCR4. The chemokine receptor CXCR4 is implicated in the metastasis of various cancers. The association of CXCR4 expression with HCC bone metastasis and patient survival was recently reported. CXCR4 expression in primary HCCs may be an independent risk factor for bone metastasis and associated with poor clinical outcome [
17]. Our transwell results indicated that depletion of CXCR4 expression resulted in significant inhibition of PVTT cell migration. These data extend the critical role of CXCR4 in promoting the migration of cancer cells. The central role of CXCR4 in cancer metastasis also raises the question of whether CXCR4 can serve as an important diagnostic target in the detection and treatment of cancer. Additionally, it is important to further establish the mechanisms that result in increased CXCR4 expression andpotentially target such pathways in cancer treatment. Thus, understanding the mechanisms that normally regulate CXCR4 expression and function should prove useful in the treatment and prevention of cancer metastasis.
Conclusions
We determined that the expression of CXCR4 in PVTT tissue was greater than that in liver cancer tissue and that the downregulation of CXCR4 by RNA interference significantly impaired the invasive ability of PVTT cells. It is possible that CXCR4 plays a critical role in the development of PVTT in HCC. The potential siRNA target we screened may have an advantageous curative effect on HCC.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NL and SQC conceived, coordinated and designed the study and contributed to the acquisition, analysis and interpretation of data and drafted the manuscript. WXG performed the experiments and were involved in drafting the article. JS and JX selected archived samples and participated in the study design and interpretation of the results. HSH participated in sample collection and data acquisition. All authors have read and approved the final manuscript.