Hepatocellular carcinoma (HCC) is the most prevalent malignant cancer of liver and is one of the major causes of tumor-related mortality, particularly in China [
Diabetes mellitus (DM) has been confirmed as one of the important risk factors of HCC in the last decade [
Insulin receptor substrate 1 (IRS-1) is an important molecule of the insulin signal transduction pathway and plays multiple varieties of biological regulations in insulin metabolism [
Two hundred and forty specimens were drawn from 140 patients, including 100 HCC tissues and 100 paracancerous (PC) tissues from 100 HCC patients, 20 liver cirrhosis (LC) tissues from 20 LC patients, and 20 chronic hepatitis (CH) tissues from 20 CH patients. These patients were treated at the China-Japan Friendship Hospital, Ministry of Health, and the Beijing Tiantan Hospital, Capital Medical University. Patients who followed these criteria were included: (1) those who had undergone surgical treatment because of tumor or other diseases and the surgical specimens could satisfy the need of scientific research and experiments; (2) those who had not been treated with any tumor-associated therapies before surgery; (3) HCC, LC, and chronic hepatitis were diagnosed by pathological examination; (4) those who had no presence of other malignancies, including lymphoma and leukemia; and (5) those who had no other severe disease, including heart, lung, brain, kidney, rheumatic, or allergic disorders.
Our research project followed strictly the principles of the Declaration of Helsinki, and it was approved by the Human Research Ethics Committee of China-Japan Friendship Hospital, Ministry of Health. Informed consent was obtained from all patients.
All the acquired surgical specimens were fixed immediately in 4% paraformaldehyde for 24 hr and transported to the laboratory for subsequent processing. They proceeded to be dehydrated and cleared in gradient ethanol and xylene, finally embedded in paraffin blocks. For pathological diagnosis, these paraffin-embedded tissues were serially sectioned and stained with hematoxylin-eosin. For immunohistochemical staining, they were cut as 4-5
Polyclonal antibody anti-HIRS-1 (No. SAB4300482-100UG), specially prepared for immunohistochemistry (IHC), was obtained from Sigma-Aldrich (Saint Louis, MO, USA). The original concentration was 1 mg/ml, and the final optimal diluted concentration was 1 : 200. The PV-6001/6002 PowerVision™ two-step histostaining reagent was a ready-to-use kit and purchased from Zhongshan Jinqiao Biotechnology Corporation (Beijing, China). Some routine reagents, including phosphate-buffered saline (PBS) buffer, citrate buffer, and Dako REAL™ EnVision™ Detection System, were also supplied by Zhongshan Jinqiao (Beijing, China). In addition, a microwave oven operating at a frequency of 2.45 GHz was used for antigen retrieval.
Five relevant indicators were recorded, including their ages, sex, infection status of hepatitis B virus (HBV) and/or hepatitis C virus (HCV), and pathologic diagnosis of HCC or others. For subsequent analysis, the age was regrouped into two groups with the cutoff of 55 years old. Pathological characteristics of the 100 HCC patients were shown, including the TNM stage, clinical classification, grade of differentiation, presence or absence of tumor thrombus of veins, location of tumors, tumor number, and the maximum diameter of tumors. For the clinical classification, three types were found in these patients, including massive type, nodular type, and small-cancer type.
Immunohistochemical (IHC) staining was strictly carried out following the measure operation steps of the manufacturer’s instructions. According to our previously published studies [
IRS-1 expression was evaluated by two researchers independently, and if they obtained inconsistent results, consultation and discussion would be used. The extent of immunostaining was scored from grade 1 to grade 4: 1, 5-25%; 2, 25-50%, 3, 50-75%; and 4, 75-100%. The staining intensity was scored from grade 1 to grade 3: 1, mild; 2, moderate; and 3, strong. The final score was the sum of two numbers, and the expression grade was determined as 2-3, weak or mild; 4-5, moderate; and 6-7, strong.
SPSS for Windows, version 21.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. The continuous variables were described as
Our study included 240 surgical specimens which were drawn from 140 patients, including 100 HCC tissues and 100 PC tissues from 100 HCC patients, 20 LC tissues from 20 LC patients, and 20 CH tissues from 20 CH patients. Table
Baseline characteristics of patients
Variables | HCC | LC | CH | |
---|---|---|---|---|
Total number, | 100 | 20 | 20 | |
Number of specimens, | 200 | 20 | 20 | |
HCC | 100 | |||
Paracancerous tissues | 100 | |||
Age, years | ||||
Mean age | 0.427 | |||
≥55, | 55 (55.0) | 8 (40.0) | 8 (40.0) | 0.276 |
<55, | 45 (45.0) | 12 (60.0) | 12 (60.0) | — |
Sex | ||||
Male, | 69 (69.0) | 13 (65.0) | 10 (50.0) | 0.262 |
Female, | 31 (31.0) | 7 (35.0) | 10 (50.0) | — |
Hepatitis virus | ||||
HBsAg+, | 66 (66.0) | 11 (55.0) | 9 (45.0) | 0.173 |
Anti-HCV+, | 7 (7.0) | 1 (5.0) | 1 (5.0) | 0.909 |
Both HBV and HCV, | 2 (2.0) | 0 (0.0) | 0 (0.0) | — |
None, | 25 (25.0) | 8 (40.0) | 10 (50.0) | 0.054 |
Pathologic diagnosis | ||||
HCC, | 100 (100) | — | — | |
Cirrhosis, | — | 20 (100) | — | |
Chronic hepatitis, | — | — | 20 (100) |
CH: chronic hepatitis; HCC: hepatocellular carcinoma; LC: liver cirrhosis
Pathological characteristics of 100 HCC patients.
Variables | 100 HCC patients |
---|---|
T stage, | Stage T1-2, 63 (63.0) |
Stage T3-4, 37 (37.0) | |
N stage, | Stage N0, 100 (100.0) |
Stage N1, 0 (0.0) | |
M stage, | Stage M0, 96 (96.0) |
Stage M1, 2 (2.0) | |
Clinical classification, | Massive type, 54 (54.0) |
Nodular type, 33 (33.0) | |
Small-cancer type, 13 (13.0) | |
Grade of differentiation, | Middle, 79 (79.0) |
High and low, 21 (21.0) | |
Tumor thrombus of veins, | Absence, 83 (83.0) |
Presence, 17 (17.0) | |
Location of tumors, | Left liver, 17 (17.0) |
Right liver, 74 (74.0) | |
Left and right liver, 9 (9.0) | |
Tumor number, | One, 61 (61.0) |
Two or more, 39 (39.0) | |
Maximum diameter of tumors, cm | 7.09 ± 3.66 |
For the TNM stage of the 100 HCC patients, 63 were defined as Stage T1 and Stage T2; no patient was found to have lymph node metastases, and only 2 patients were identified as Stage M1. The mean maximum diameter of tumors was
Expression of IRS-1 was examined by IHC staining, and Table
Expression of insulin receptor substrate 1 (IRS-1) in human HCC.
Tissues/protein | IRS-1 expression | |||
---|---|---|---|---|
Negative | Weak | Moderate | Strong | |
Tissues of detection | ||||
HCC, | 59 (59.0) | 15 (15.0) | 18 (18.0) | 8 (8.0) |
PCT, | 83 (83.0) | 11 (11.0) | 6 (6.0) | 0 (0.0) |
LC, | 17 (85.0) | 2 (10.0) | 1 (5.0) | 0 (0.0) |
CHT, | 18 (90.0) | 2 (10.0) | 0 (0.0) | 0 (0.0) |
HCC | <0.001 | 0.400 | 0.009 | — |
HCC | 0.028 | 0.815 | 0.263 | — |
HCC | 0.008 | 0.815 | — | — |
PCT | 1.000 | 1.000 | 1.000 | — |
PCT | 0.655 | 1.000 | — | — |
LC | 1.000 | 1.000 | — | — |
CHT: chronic hepatitis tissues; HCC: hepatocellular carcinoma; LC: liver cirrhosis; PCT: paracancerous tissues,
Expression of IRS-1 by immunohistochemical analysis in human hepatocellular carcinoma (HCC) tissues. (a-h) Immunohistochemical staining; (c, f) hematoxylin-eosin staining. Bars:
The 100 paracancerous tissues specimens were drawn from the same group patients, and they had a one-to-one relationship with the 100 HCC samples. Figures
IRS-1 expression by immunohistochemistry in paracancerous tissues (a, b), liver cirrhosis (c, d), and chronic hepatitis tissues (e, f). (a, c, e) Hematoxylin-eosin staining; (b, d, f) immunohistochemical staining. Bars:
For the 20 LC patients and 20 CH patients, IRS-1-positive cells were observed in five patients (Figures
The Pearson chi-square test was used to compare the expression intensity of protein IRS-1 in human HCC with other tissues. For the total positive rate of IRS-1 expression, a significantly increased level was observed in human HCC (Figure
Proportions with the total positive expression (a), weak positive (b), moderate positive (c) and strong positive expression (d) of IRS-1 in four group tissue samples, including hepatocellular carcinoma (HCC), paracancerous tissues (PCT), liver cirrhosis (LC), and chronic hepatitis tissues (CHT). The
The 140 patients were redivided into two groups based on the expression status of protein IRS-1 to determine the association of baseline characteristics with IRS-1 expression (Table
Univariate and multivariate analyses for the association of baseline characteristics with IRS-1 expression in total 140 patients.
Variables | IRS-1 expression | Univariate | Multivariate analysis† | |||
---|---|---|---|---|---|---|
Positive | Negative | Adjusted OR | 95% CI | |||
Age (years), | ||||||
≥55 | 25 (35.2) | 46 (64.8) | 0.547 | 1.121 | 0.509-2.470 | 0.776 |
<55 | 21 (30.4) | 48 (69.6) | — | |||
Sex, | ||||||
Male | 34 (37.0) | 58 (63.0) | 0.153 | 1.818 | 0.778-4.247 | 0.167 |
Female | 12 (25.0) | 36 (75.0) | — | |||
HBV, | ||||||
Positive | 26 (29.5) | 62 (70.5) | 0.278 | 0.390 | 0.169-0.904 | 0.028 |
Negative | 20 (38.5) | 32 (61.5) | — | |||
HCV, | ||||||
Positive | 2 (18.2) | 9 (81.8) | 0.456 | 0.215 | 0.040-1.160 | 0.074 |
Negative | 44 (34.1) | 85 (65.9) | — | |||
HCC patients, | ||||||
HCC | 41 (41.0) | 59 (59.0) | 0.001 | 6.60 | 2.243-19.425 | 0.001 |
Non-HCC | 5 (12.5) | 35 (87.5) | — |
CI: confidence interval; HBV: hepatitis B virus; HCC: hepatocellular carcinoma; HCV; hepatitis C virus; OR: odds ratio,
Binary logistic regression model calculation was used to perform the multivariate analysis. Considering the results of univariate analysis and study population, five variables were entered, including the age, sex, infection status of HBV and/or HCV, and pathologic diagnosis of HCC or others. We found that significant differences were observed for the presence of HBV infection (OR 0.390, 95% CI 0.169-0.904,
Diabetes has been identified as one risk factor for HCC, and IRS-1 is an important molecule of the insulin signal transduction pathway of DM. Based on these theoretical foundations, our study was designed to determine the expression and significance of IRS-1 in human HCC. PC, LC, and CH tissues were served as control subjects, and we found that the positive expression of IRS-1 in HCC was increased significantly. In addition, we observed that eight of the total 240 specimens had the strong signals of IRS-1-positive expression and all of them were HCC tissues. Moreover, multivariate analysis supported this conclusion and showed that IRS-1 was independently associated with the diagnosis of HCC and the value of adjusted OR was 6.60.
Our results indicated that increased IRS-1-positive expression may play an important role in the occurrence and development of human HCC. Some efforts have been made to gain these relatively reliable results. The first was that 200 HCC and paracancerous tissues were included in our examinations and they had a one-to-one relationship with each other. Such a comparison was much better than the operation in which HCC and PCT were drawn from different patients. The second was that 40 LC and CH patients were also served as the control subjects. Coincidentally, the pathological diagnosis of PCT was divided into two types, namely, LC and CH. Therefore, these two groups of specimens could be regarded as the controls either for HCC or for PCT.
Our conclusion that positive expression of IRS-1 was increased significantly in HCC was supported by some of the previously published studies [
However, there is also a different voice, such as another research which was conducted to detect the expression of human IRS-1 and insulin signaling proteins under different pathophysiologic conditions of hepatic disease [
Our study included 240 surgical specimens drawn from 140 patients and found that the IRS-1 expression was increased significantly in HCC. We also found that 33 of the 240 specimens were moderate or strong positive of IRS-1 expression, and 78.8% of the 33 specimens were HCC. Only eight were strong positive, and all of them were HCC. Considering that PCT can only be diagnosed as LC or CH in our study, the 140 non-HCC specimens were divided into two subgroups, namely, LC and CHT. For the IRS-1 expression, subgroup analysis was performed and no significantly statistical differences were observed. These further analyses supported our final conclusion.
Fortunately, some studies have investigated the molecular mechanisms of the effect of IRS-1 overexpression on the occurrence and development of HCC and their results supported our conclusion [
In addition, in our study, we found that the status of HBV infection was associated with the positive expression of protein IRS-1 (Table
Some limitations should be acknowledged. The first was that all the patients were drawn from one medical center of China and all the specimens were obtained from the surgery operation. Therefore, bias selection could not be avoided and we set up some different kinds of controls to ensure the accuracy of the results. The second was that the expression was examined simply by IHC and more techniques were required.
In conclusion, our study found that IRS-1-positive expression in HCC was increased significantly and may play an important role in the development of human HCC. For a better understanding of the molecular mechanisms, more studies are required.
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
The authors declare that there is no conflict of interest.
The authors would like to thank Professor Shu-Kun Yao, China-Japan Friendship Hospital, for some of his guidance in the design and implementation of this research. This work was supported by Z131107000413067 from the Beijing NOVA Programme, and 2014-QNYC-B-01, 2013-QN-07, and 2013-QN-06 from the Research Fund of the China-Japan Friendship Hospital, Ministry of Health.