The correlation of NLRC3 expression with the progression and prognosis of hepatocellular carcinoma

Highlights

• Hepatocellular cancer tissues have reduced NLRC3 expression.

• NLRC3 expression inversely correlated with tumor progression and prognosis.

• NLRC3 promotes proliferation and inhibits apoptosis in hepatocellular cancer cells.

Summary

NLRC3 is a member of the nucleotide-binding domain and leucine-rich repeat (NLR) family protein that plays a role in inflammation and immunity. Although chronic inflammation has been identified as a hallmark of cancer, NLRC3 expression correlation with the development and prognosis of hepatocellular carcinoma (HCC) is unclear. In the present study, we first used Oncomine and OncoLnc database to determine the clinical significance of NLRC3 in HCC. Then we performed quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical staining (IHC) and analyzed the correlation between NLRC3 expression and clinicopathological features of HCC in a Chinese population. We found that high levels of NLRC3 messenger RNA (mRNA) correlated with a favorable clinical outcome; furthermore, expression of NLRC3 was significantly reduced in the cancer tissue in patients compared with noncancerous hepatic tissues. NLRC3 reduction was correlated with Edmondson grade and metastasis. Kaplan-Meier survival analysis revealed that HCC patients with high expression of NLRC3 have a more favorable prognosis compared with those with low expression of NLRC3. We then used short hairpin RNA to knock down NLRC3 expression in HCC cell lines and evaluated its effect on cell proliferation and apoptosis. Suppression of NLRC3 expression promoted cell proliferation and inhibited apoptosis in vitro. Genomic analysis of the OncoLnc database also showed that NLRC3 mRNA level was directly correlated with mRNA levels of inflammasome components caspase-1, IL-1β, and IL-18. Based on our present study, down-regulated expression of NLRC3 may play an important role in cancer progression and prognosis of HCC by acting as a tumor suppressor.

Introduction

Hepatocellular carcinoma (HCC) accounts for 85% to 90% of primary liver cancers, which is one of the most common causes of cancer-related deaths worldwide [1]. Although advanced modern medicine promotes the development of techniques for the diagnosis and therapy of HCC [2], [3], the prognosis and survival of HCC patients remain disappointing because of high rate of recurrence and metastasis [4], [5]. Particularly in China, it accounts for nearly 55% of all HCC cases in the world [6] owing to high rate of chronic hepatitis B virus (HBV) infection [7]. Although many molecular biomarkers involved in HCC have been identified, the mechanism remains unclear.

Inflammation, a hallmark of cancer, is considered to play critical roles during different stages of tumor development, such as initiation, promotion, malignant conversion, invasion, and metastasis [8]. Up to 80% of HCCs occur in the course of chronic liver diseases, such as hepatitis or alcoholic or nonalcoholic steatohepatitis [9]. Chronic inflammation is associated with DNA and tissue damage, including genetic and epigenetic changes that may lead to tumorigenesis, whereas acute inflammation prevents pathogenic infection. Furthermore, studies revealed that proinflammatory cytokines presented in peritumoral tissues or systemic circulation associated with recurrence and poor prognosis of HCC [10], [11]. The nucleotide-binding, leucine-rich repeat-containing (NLR) protein family is an intracellular sensor that has many functions, including inflammatory and anti-inflammatory roles [12], [13]. The NLRC3 (NLR family with a caspase activation and recruitment domain 3) is a member of the NLR family proteins that functions as a negative regulator of inflammatory signaling pathways. Previous studies have shown that NLRC3 is activated by Toll-like receptors and the DNA sensor STING in response to pathogen-associated molecular patterns or to virus infection [14], [15]. One of the most important functions of the NLR family is to serve as central components of an inflammasome, and inflammasome was reported to act as a strategic protein complex, providing a molecular platform initiating signaling cascades of inflammatory events [16]. Gültekin et al [17] also showed that elevated expression of NLRC3 may act as an anti-inflammatory cytosolic protein. Liu et al [18] revealed that the expression level of NLRC3 in colorectal cancer (CRC) was significantly lowered in CRC than in healthy controls; furthermore, the extent of NLRC3 gene reduction was correlated with cancer progression. Karki et al [19] showed that NLRC3 protects against the development of CRC through the inhibition of the PI3K-mTOR pathway.

However, the physiological role of NLRC3 in HCC has remained largely unknown. In the present study, we performed analysis of NLRC3 expression in human HCC by combining the bioinformatics analysis of publicly available databases. Then we confirmed the role of NLRC3 in 330 cases of HCC using immunohistochemical staining. Furthermore, we also investigated the biological function of NLRC3 in vitro by short hairpin RNA (shRNA). Based on our present study and this broad analysis, NLRC3 may represent valuable candidate biomarkers for HCC prognosis and treatment.