Background

Hepatocellular carcinoma (HCC) is one of the most malignant tumors with a poor prognosis. One reason for this is that the pathogenesis of HCC is complex, especially considering hepatitis B-induced liver cirrhosis (HBILC) is the primary pathological basis of HCC. In the process of evolution to HCC, coupled with the role of the hepatitis B virus (HBV), some proteins and signal pathways closely related to inflammation and tumors play an important regulatory role. Our research team has found that macrophage migration inhibitor (MIF) is gradually over-expressed in chronic hepatitis B, HBILC, and HCC,1,2 suggesting that MIF may play a crucial role in the evolution of chronic hepatitis B to cirrhosis and HCC. According to the literature, MIF, an inflammatory factor, plays a role in promoting inflammation and carcinogenesis. This is closely related to the activation of related signal pathways,3–6 while the signaling pathway of the extracellular-regulated kinases 1 and 2 (ERK1/2), an important sub-pathway of the mitogen-activated protein kinase (MAPK) signaling pathway, is closely related to the occurrence and development of a variety of tumors. For example, the up-regulation of ERK1/2 expression can promote the formation and development of gastric cancer, melanoma, esophageal cancer, and colon cancer.7–10 Many studies have also reported that abnormal activation of the ERK1/2 signaling pathway is closely related to HCC.11,12 However, there is no apparent research on whether a correlation exists between the ERK1/2 signaling pathway and MIF at the tissue level. Therefore, the research team collected normal liver tissues, HBILC tissues, and HCC tissues to detect the expression of proteins and nucleic acids of MIF and ERK1/2, as well as phosphorylated ERK1/2 protein, to understand the correlation between these two proteins and the occurrence and development of HBILC and HCC.

Subjects and Methods Specimen Collection and Preparation

Forty-eight HCC tissue specimens with HBILC and corresponding cirrhosis tissues 5 cm away from HCC were collected as the experimental group. Tissue specimens were collected from 36 male and 12 female patients with ages ranging from 35–65 years old and an average age of 41.05 ± 3.81 years old. Normal liver tissues from 20 patients with gallstones or hemangiomas were collected as the control group. The diagnostic criteria of all HCC patients were based on the Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022 version), which was revised and updated again by the National Health Commission in 2022.13 All tissue specimens were fixed with 10% formaldehyde, embedded in paraffin, and made into paraffin sections of tissue chip with a section thickness of about 4 µm.

Experimental Reagents and Methods

The MIF monoclonal antibody was procured from Santa Cruz (USA) with a dilution ratio of 1:80, the monoclonal antibody of Rabbit Anti-human Thymocyte Immunoglobulin ERK1/2 and the goat anti-rabbit secondary antibody were procured from Abcam (UK) with a dilution ratio of 1:80, and the phosphorylated rabbit anti-human thymocyte immunoglobulin ERK1/2 monoclonal antibody was procured from CST (USA) with a dilution ratio of 1:100. MIF, ERK1/2 in situ hybridization kit (person) was procured from Roche (Shanghai). Immunohistochemical technique was used for detecting the expression of MIF, ERK1/2 protein and phosphorylated ERK1/2 protein in the liver tissues of the experimental group and the control group. After adding the first and second antibodies, they had been incubated at 37°C for 50 min. The expression of MIF and ERK1/2 nucleic acids in the experimental group and the control group was detected by in situ hybridization. In this step, 20% hydrogen peroxide was added to inactivate the activity of endogenous peroxidase. It was necessary to incubate the MIF and ERK1/2 nucleic acids in a 37°C incubator for 10 min, expose the MIF and ERK1/2 nucleic acids in a 37°C incubator for 20 min, and then fix the 37°C incubator for 10 min, pre-hybridize in a 37°C incubator for 4h, hybridize in a 37°C incubator for 24h, and then add the sealing solution for 30 min, the biotinylated rabbit anti-digoxin was incubated at 37°C for 1h, and SABC and biotinylated peroxidase were incubated at 37°C for 30 min respectively.

Result Interpretation Criteria

The protein is mainly expressed in the cytoplasm, while the phosphorylated protein is primarily expressed in the nucleus. The presence of brownish-yellow particles indicates a positive result. Figures were read at high magnification (× 400). For each section, a non-repeating field was chosen in the upper, middle, lower, left, and right sites. Both the percentage and staining intensity of positive cells among all cells of the same type were observed. Results were calculated based on the combination of these two factors. The scoring standard for the percentage of positive cells was as follows: <10% recorded as 0 points, 10–40% recorded as 1 point, 41–70% recorded as 2 points, and >70% recorded as 3 points. The scoring standard for staining intensity was as follows: 0 points for no visible staining, 1 point for light yellow, 2 points for brownish-yellow, and 3 points for yellow-brown. A comprehensive score was calculated from the sum of the two scores, where cells with a score ≤2 points were negative and cells with a score >2 points were positive.

Statistical Methods

The data were processed and analyzed using SPSS 17.0. Count data were evaluated using Pearson’s X2 test, and P<0.05 was considered statistically significant.

Results Expression of MIF and ERK1/2 Proteins

The positive expression of MIF protein was mainly in the cytoplasm, which was highly expressed in tissues of HCC and HBILC, while the positive rate in normal liver tissue was low (Figure 1A–C). The positive expression of ERK1/2 protein was primarily in the cell membrane and cytoplasm; MIF expression in the HCC and HBILC groups was also higher than that in the control group (Figure 2A–C). There were significant differences in MIF and ERK1/2 protein expression in different liver tissues between all groups (P<0.05). However, there was no significant difference between the HCC group and the HBILC groups (P>0.05; Table 1).

Table 1 Expressions of MIF and ERK1/2 Proteins in Experimental Groups and Control Group

Figure 1 Expression of MIF. (A) Positive expression of MIF in HCC×200. (B) Positive expression of MIF in HBV-LC×200. (C) Negative expression of MIF in normal liver tissue×200.

Figure 2 Expression of ERK1/2. (A) Positive expression of ERK1/2 in HCC×200. (B) Positive expression of ERK1/2 in HBV-LC ×200. (C) Negative expression of ERK1/2 in normal liver tissue×200.

Expression of MIF and ERK1/2 Nucleic Acids

MIF mRNA and ERK1/2 mRNA were mostly positively expressed (present) in the HCC group and HBILC group and negatively expressed (absent) in the control group (Figures 3A and 4C); that is, there were significant differences in the expression of the two nucleic acids between the HCC group and the control group and between the HBILC group and the control group (P<0.05). There were no significant differences in the expressions of the two nucleic acids between the HCC and HBILC groups (P>0.05, Table 2).

Table 2 Expressions of MIF and ERK1/2 Nucleic Acids in Experimental Groups and Control Group

Figure 3 Expression of MIFmRNA. (A) Positive expression of MIFmRNA in HCC×200. (B) Positive expression of MIFmRNA in HBV-LC×200. (C) Negative expression of MIFmRNA in normal liver tissue×200.

Figure 4 Expression of ERK1/2 mRNA. (A) Positive expression of ERK1/2 mRNA in HCC×200. (B) Positive expression of ERK1/2 mRNA in HBV-LC ×200. (C) Negative expression of ERK1/2 mRNA in normal liver tissue×200.

Expression of PERK1/2

The expression of pERK1/2 was localized in the nucleus but was also detected in the cell membrane and cytoplasm. The present study revealed that pERK1/2 had a high expression rate in the HCC group and a low expression rate in the HBILC and control groups (Figure 5A–C). There was a significant difference between the HCC group and HBILC group and between the HCC group and control group (P<0.05). However, there was no significant difference between the HBILC and control groups (P>0.05, Table 3).

Table 3 Expression of Phosphorylated ERK1/2

Figure 5 Expression of pERK1/2. (A) Positive expression of pERK1/2 in HCC×200. (B) Suspected positive expression of pERK1/2 in HBV-LC ×200. (C) Negative expression of pERK1/2 in normal liver tissue×200.

Discussion

At present, the global incidence rate of HCC is still mounting. Although various methods have played a role in the treatment of HCC—especially the development and clinical application of molecular targeting and immune checkpoint inhibitors—and improved the curative effects of HCC to a certain extent, they are not successful in all patients. One reason is that the pathogenesis of HCC is extremely complex. During the evolution from HBILC to HCC, HBV-induced hepatocyte inflammation14 and abnormal expressions of some genes, proteins, and cytokines (such as the abnormalities of signaling pathways related to proteins regulated by cytokines) is one of the mechanisms of HCC occurrence and development. MIF is a common one and is mainly derived from T lymphocytes, which are secreted to the outside of cells in the form of autocrine and paracrine and play a role by binding with membrane receptors.15 MIF is a pleiotropic cytokine with pro-inflammatory and carcinogenic effects16,17 and is not only related to the occurrence of many inflammatory diseases18–20 but also participates in the occurrence and development of a variety of tumors21–24 and plays a role in the progression of HBILC.25,26 The MAPK signaling pathway is the main pathway of intracellular signal transduction and is the core link of tumor cell proliferation, differentiation, apoptosis, and migration. It consists of four subfamilies: extracellular signal-regulated kinase (ERK), p38 kinase (p38 MAPK), c-Jun terminal kinase (JNK), and extracellular signal-regulated kinase 5 (ERK5). After extracellular stimulation by cytokines, growth factors, and other elements, related pathway proteins are phosphorylated and transmitted into cells and nuclei through a typical tertiary kinase cascade to activate many transcription factors. Then they regulate many physiological processes such as cell proliferation, differentiation, and apoptosis, as well as promoting tumor growth, invasion, and migration. In recent years, several studies suggest that the occurrence of HBILC and HCC is related to the abnormalities of ERK1/2, p38 MAPK, and JNK1 sub-pathways.11,27–29 This suggests that the occurrence and development of HBILC and HCC are closely related to the abnormalities of MIF, ERK1/2, p38 MAPK, and JNK1.

In the early stage of this study, it was found that hepatocellular carcinoma and its adjacent tissues highly expressed MIF and ERK1/2, suggesting that the occurrence of hepatocellular carcinoma is closely related to the expression of these two proteins.30 However, because there may be satellite lesions of cancer in the adjacent tissues of hepatocellular carcinoma, which is different from the simple liver cirrhosis tissues, and liver cirrhosis is the pathological basis of the occurrence of hepatocellular carcinoma. Therefore, to further explore the correlation between the expression of MIF and ERK1/2 in the process of hepatocarcinogenesis, HBV-related hepatocellular carcinoma and liver cirrhosis tissues were collected in this study and expression of these two proteins and nucleic acids were detected. The present study revealed that the protein and nucleic acid expressions of MIF and ERK1/2 were significantly increased in both the HBILC and HCC groups, and the differences compared with the control group were statistically significant (P<0.05 for both). However, there was no significant difference between the HCC group and the HBILC group (P>0.05, Figures 1A and 4C, Tables 1–2). The expression of pERK1/2 was increased in the HCC group, which was found primarily in the nucleus. There was a significant difference between the HCC and HBILC groups and between the HCC and control groups (P<0.05), but there was no significant difference between the HBILC group and the control group (P>0.05, Figure 5A–C and Table 3). These results suggest that the abnormal expressions of MIF and ERK1/2 are closely related to the occurrence and development of HBILC and HCC. Abnormal expression of pERK1/2 could be observed in the nuclei of HCC but not in the nuclei of HBILC, suggesting that ERK1/2 phosphorylation may be one of the mechanisms leading to HCC. Meanwhile, MIF may play an important regulatory role, for which the specific mechanism is worthy of further in-depth research.

Data Sharing Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

This study was conducted with approval from the Ethics Committee of the 940 Hospital of Joint Logistic Support Force of People’s Liberation Army. This study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from all participants.

Acknowledgment

As one of the members of this study group, some of the pathological specimens involved in Yu Haipeng’s study30 were sourced from the 940th Hospital of the Joint Logistics Support Force of Chinese People’s Liberation Army (normal liver tissues, hepatocellular carcinoma tissues and paracancerous tissues), and the others came from the Cancer Hospital affiliated to Tianjin Medical University Cancer Institute & Hospital (normal liver tissues, hepatocellular carcinoma tissues and paracancerous tissues). In his study, he first detected the expression of MIF, ERK1/2 and p-ERK1/2 in normal liver tissues, hepatocellular carcinoma and paracancerous tissues, as well as the expression of MIFmRNA and ERK1/2 mRNA. He found that the expression of these three proteins and two nucleic acids in hepatocellular carcinoma and paracancerous tissues was significantly higher than that in normal liver tissues, and then focused on adding the recombinant gene MIF (rMIF) to liver cancer cells and normal liver cells. He used Western and RT-PCR methods to detect the expression of ERK1/2, p-ERK1/2 and ERK1/2 mRNA in these two cells. The results showed that with the increase of rMIF concentration, the expression of ERK1/2, p-ERK1/2 and ERK1/2 mRNA was significantly higher than that of normal hepatocytes. The conclusion, MIF may promote the occurrence and development of hepatocellular carcinoma through ERK1/2 signal pathway.

Dr Xiao-Hui Yu is also a member of this subject, and the pathological samples involved in the study were all sourced from the 940th Hospital of the Joint Logistics Support Force of Chinese People’s Liberation Army (normal liver tissues, liver cirrhosis tissues and hepatocellular carcinoma tissues). The study mainly focuses on detecting the expression of three proteins (MIF, ERK1/2 and p-ERK1/2) and two nucleic acids (MIF mRNA and ERK1/2 mRNA) in normal liver, liver cirrhosis and hepatocellular carcinoma tissues. The results showed that the protein and nucleic acid of MIF and ERK1/2 were low expressed in normal liver group, high expressed in cirrhosis group and HCC group, while pERK1/2 was low expressed in normal liver tissues and cirrhosis group, and high expressed in HCC group. The expression of pERK1/2 in HCC group was significantly different from that in cirrhosis group and normal control group (P < 0.05), the expression of pERK1/2 in the cirrhosis group is not significantly different from the normal expression in the normal liver tissues control group (P > 0.05). Thus, the low expression of pERK1/2 in cirrhosis group of this study is different from the high expression in hepatocellular carcinoma tissues of Yu Haipeng, suggesting that the occurrence and development of cirrhosis and hepatocellular carcinoma are not only related to the high expression of MIF, but also may be related to the activation of ERK1/2, a key protein, especially the carcinogenesis of liver cirrhosis is more closely related to the phosphorylation of ERK1/2. Therefore, the study focused on the relationship between the occurrence of canceration of liver cirrhosis and the phosphorylation of ERK1/2.

Funding

This study was funded by the Science and Technology Plan of Gansu Province (Key research and development Programmes-Social Development) (No.20YF8FA099).

Disclosure

The authors report no conflicts of interest in this work.

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