The RPL28 gene is the typical gene which encodes ribosomal protein with many pseudo-genes scattered throughout the genome and different splicing transcription forms. In this study, the expression of RPL28 gene was found to be significantly increased in sorafenib resistant HepG2 cells compared with parental HepG2 cells. Ribosomes act as a processing factory of protein polypeptide chains and the expression of RP itself reflects the state of cell growth. Ribosome proliferation also indicates proliferative activity of both normal and tumor tissues. Growing cells have more efficient ability for translating RP mRNA than stationary cells. Thus, cells with proliferating activity usually have high RP levels. Therefore, when HepG2 cells developed resistance to sorafenib, the cell proliferation inhibited due to drug action became active again, and the expression of ribosomal protein increased accordingly, which could explain our experimental results.
In order to verify the experimental results, we constructed a RPL28 RNA interference lentiviral vector, and the HepG2 sorafenib resistant cells were infected with the lentivirus to down-regulate the RPL28 gene expression. It was found that knocked out of RPL28 gene significantly inhibited the proliferation of HepG2 sorafenib resistant cells and increased apoptotic cells. Our results suggested that interfering RPL28 gene could inhibit the proliferation of HepG2 sorafenib resistant cells, reversing sorafenib resistance. Therefore, up-regulation of RPL28 gene might be one of the causes of sorafenib resistance, and its mechanism is associated with the effect of RP in tumors. Changes in RP expression may affect the protein translation process. Both the assembly and function of ribosomes depend on the balance of the quantity of RP. The increased ribosomes may accelerate the translation of ribosomes and affect the synthesis of proteins that affect the metabolism and biological functions of cells. We speculated that the increased RPL28 gene expression would accelerate mRNA translation and protein synthesis, promote HepG2 cell proliferation, and lead to sorafenib resistance.
Ribosomal proteins can regulate cell apoptosis. Zhang et al. reported that RP S15A was highly expressed in glioma compared with normal tissues. Down-regulation of RPS15A could induce apoptosis of glioma cells . Our results indicated that the increased expression of RPL28 gene might down-regulate certain apoptotic proteins, inhibit the programmed cell death of HepG2 cells, and result in sorafenib resistance. The most reported function of ribosomal protein is related to tumor suppressor gene P53. Peng et al. reported that ribosomal protein L23 inhibit P53 ubiquitination by negatively regulating murine double minute 2 (MDM2), thereby activating P53 to inhibit tumor growth [17, 18]. It might be possible that the increased expression of RPL28 gene also regulate the P53 gene, and lead to sorafenib resistance.
Morris hepatoma3924A is a highly metastatic cell strain of rat liver cancer. Due to its high tumor formation rate in vivo, it has been widely used to establish various animal models. Jun et al. applied Morris hepatoma3924A in ACI rat to explore nanoscale integrin combined with arterial chemoembolization for the treatment of liver carcinoma . In this study, Morris hepatoma3924A in ACI rat model was used to study sorafenib resistance in vivo. It was found that the tumor growth was effectively controlled in the sorafenib sensitive stage. The tumor had extensive metastasis in the abdominal cavity in the sorafenib resistant stage.
Real time qPCR and IHC results showed that the protein expression of RPL28 gene increased in the resistant phase compared with the sensitive phase, which is in consistent with the results in vitro, and indicating that RPL28 gene might be the sorafenib-resistant gene in vivo. In addition, immunohistochemical staining results showed that Ki-67 protein expression increased in the drug-resistant stage compared to the sensitive stage. Ki-67 is used to judge cell proliferative activity. It is expressed in all cell cycles (G1, S, G2 and M stages), but not expressed in G0 stage. Previous studies indicated that the KI-67 expression is closely associated with cell proliferation, differentiation, invasion and metastasis, and prognosis of various tumors. In this study, ki-67 protein expression increased in sorafenib resistance rats, suggesting that sorafenib resistance is related to cell proliferation. The cell cycle regulatory core molecule is CDK, which is dependent on the expression of Cyclin. CDK4 is one of the CDK series of protein kinases. CDK4 kinase activity is activated when CDK4 binds to periodic protein D1 (cyclin D1) to form the CDK4 / cyclin D1 complex, which phosphorylates Retinoblastoma protein (Rb). Rb protein releases a transcription factor E2F, results in the expression of S phase related proteins induced by E2F. It promotes cells to complete DNA replication, quickly across the G1 to S phase limit point, stimulate cell growth and division . Over-expression of CDK4 caused HepG2 cells growth out of control, promoted cell proliferation and abnormal differentiation leading to sorafenib resistance. Bcl-2 (B-cell lymphoma 2 gene) is an oncogene. Its family of proteins play an important role in mitochondrial apoptosis. The Bcl-2 protein family has high homology and contains BH (1–4) conserved domains. The function of Bcl-2 protein family can be divided into two categories, one is anti-apoptotic: including Bcl-2, Bcl-XL and McL-1, that are located in the mitochondrial outer membrane, endoplasmic reticulum and nuclear membrane. The other is apoptotic promoting, including Bax, Bak and Bid. They are mostly located in the cytoplasm .