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Hepatocellular carcinoma (HCC), often referred to as 'the king of cancer,' is the primary malignancy of the liver. It typically arises in individuals with chronic liver disease caused by hepatitis virus infection. HCC frequently develops in patients with HCV-related liver cirrhosis, with an annual incidence rate of approximately 3% (De Re et al., 2019). The World Health Organization estimates that 130-170 million people worldwide are infected with hepatitis C virus (HCV), leading to over 350,000 deaths annually due to HCV-related liver diseases (Coppola et al., 2015).
The tumor suppressor gene TP53 is located on chromosome 17 and encodes a 53-kDa nuclear phosphoprotein.
TP53 activation leads to the expression of p21, which influences the G1 phase checkpoints of the cell cycle, regulates cell entry into the S phase, and manages the repair of damaged cells. When the repair process fails, apoptosis is induced to prevent carcinogenesis (Insinga et al., 2013). Mutations in the TP53 gene are widely associated with cancer. In addition to mutations, a common G/C single nucleotide polymorphism occurs in exon 4, codon 72 of the TP53 gene.
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This SNP results in the substitution of guanine (CGC) with cytosine (CCC) nucleotide, leading to the production of proline (Pro) instead of arginine (Arg) at the affected codon. This SNP is known as TP53 Arg72Pro and affects TP53 function (Buchman et al., 1988). Each individual inherits two alleles of the TP53 gene, one from each parent, determining the TP53 genotype, which can be heterozygous polymorphism (Arg/Pro), homozygous with no polymorphism (Arg/Arg), or homozygous polymorphism (Pro/Pro).
These two polymorphic variants of TP53 have structural differences and exhibit distinct biochemical and biological properties (Siddique and Sabapathy, 2006).
Blood samples were collected from the study participants, including twenty patients with chronic hepatitis C, forty patients with HCV-related liver cirrhosis, twenty patients with HCV-related liver cirrhosis with hepatocellular carcinoma (HCC), and twenty healthy adults matched for age and sex, who served as the control group. Each participant underwent a comprehensive medical history assessment and clinical examination. The following tests and examinations were conducted for all patients: liver function tests, hepatitis markers, Quantitative HCV PCR, measurement of Alpha-fetoprotein (AFP) levels, and abdominal ultrasound. Patients with hepatic focal lesions underwent triphasic Abdominal CT or MRI to confirm the diagnosis of HCC. Informed written consent was obtained from all participants.
Genomic DNA was extracted from the Buffy coat collected from EDTA blood using the Thermo Scientific GeneJET Whole Blood Genomic DNA Purification Mini Kit, following the manufacturer's instructions. The extracted DNA was stored at -20°C for subsequent use in the amplification of the target gene by PCR.
PCR assays were performed using 200 ng of the extracted DNA in a total reaction volume of 50 μL. The PCR mix consisted of 25 μL of Master mix (MyTaq Red Mix, Bioline, England), 2.5 μL of each primer ('forward': 5'-TGAGGACCTGGTCCTCTGACT-3' and 'reverse': 5'-AAGAGGAATCCCAAAGTTCCA-3'). Thirty-five cycles of amplification were carried out in a thermal cycler (T Gradient - Biometra). The PCR protocol included an initial denaturation of DNA at 95°C for 7 minutes, followed by each cycle comprising a denaturation step at 94°C for 60 seconds, primer annealing at 50°C for 60 seconds, an extension step at 72°C for 45 seconds, and a final extension step at 72°C for 10 minutes following the last cycle. The amplified products were visualized on a 2% agarose gel stained with ethidium bromide. The stained gels were visualized and documented using a gel documentation system and analyzed visually to determine the size of the PCR amplicon of the target gene, which was 416 bp.
The amplified products obtained from PCR were subjected to digestion using the BstUI restriction endonuclease. In each restriction digestion reaction, 21.5 μL of the amplified PCR product was digested with 1 μL of BstUI restriction enzyme and 2.5 μL of 1X restriction enzyme buffer. The reaction mixture (25 μL) was incubated at 37°C for 1 hour. Subsequently, the digested fragments were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized under UV illumination using a gel documentation system. The size of DNA fragments was determined by direct comparison with a 100 bp DNA ladder (Jena Bioscience, Germany). The restriction fragments generated after digesting the target gene with BstUI restriction endonuclease are presented in Table 1.
| Size of PCR Amplicon | Size of Restriction Fragments | Type of Polymorphism | Genotype/Allele |
|---|---|---|---|
| 416 bp | 416 bp | No polymorphism | Arg/Arg |
| 263, 161 bp | Pro/Pro | Homozygous polymorphism | C/C |
| 416, 263, 161 bp | Arg/Pro | Heterozygous polymorphism | G/C |
After centrifugation, blood samples were separated to obtain serum. The concentration of human TP53 in serum samples was determined using the ELISA Kit (Sinogeneclon Biotech, Catalog No. SG-10350) and a double antibody technique, following the manufacturer's instructions. The absorbance of the samples was measured at a wavelength of 450 nm. The concentration of TP53 in the samples was determined by comparing the optical density (O.D.) of the samples to the standard curve.
Data analysis was performed using IBM SPSS Statistics for Windows, version 25. Quantitative data were presented as mean ± standard deviation, median, and range, while qualitative data were expressed as numbers and percentages. Independent Samples t-test and One-Way ANOVA test were utilized for normally distributed data. Chi-square (χ2) test and Fisher's Exact Test were employed for the comparison of qualitative variables as appropriate. Univariate and multivariate binary logistic regression analyses were conducted to identify predictor variables of HCC. A significance level of 5% was chosen for all statistical tests conducted in the study.
This study included a total of 80 patients, comprising 46 males and 34 females. Among them, 40 patients had HCV-related liver cirrhosis, with a mean age of 56.45 ± 9.18 years (range: 33-75 years), including 22 males (55%). The remaining 20 patients had HCC on top of HCV-related liver cirrhosis, with a mean age of 59.75 ± 8.36 years (range: 42-75 years), including 14 males (70%). Additionally, 20 patients had HCV without liver cirrhosis, with a mean age of 39.1 ± 11.2 years (range: 22-60 years), including 10 males (50%). Twenty healthy controls, matched for age and sex with the patient groups, were also included in the study.
PCR-RFLP results revealed the distribution of TP53 genotypes among the study groups, as summarized in Table 2. The GG (wild type) genotype was most commonly observed in the LC group (55%), CHCV group (45%), and the control group (55%). In contrast, the CC genotype was detected at a higher frequency in the HCC group (40%), followed by the LC group (22.5%). A statistical analysis comparing the distribution of the CC genotype across the four study groups indicated that patients with HCC had a significantly higher frequency of the CC (pro/pro) genotype compared to the other groups.
The allele frequencies among the four groups are detailed in Table 2. The G allele was more frequently detected in the control group (72.5%), followed by the CHCV group (67.5%) and LC group (66.3%). In contrast, the C allele was most common in the HCC group (55%). Consequently, the allelic frequencies in the HCC group were significantly different from those in the other study groups (P = 0.002).
The GG genotype and G allele exhibited the highest frequency in the control group, while the CC genotype and C allele were more frequently detected in the HCC group.
| Parameters | Groups | HCC (n=20) | LC (n=40) | CHCV (n=20) | Control (n=20) | P-value | |
|---|---|---|---|---|---|---|---|
| Distribution of Genotypes | Homozygotes (Wild type) (arg/arg) | GG | 6 (30%) | 22 (55%) | 9 (45%) | 11 (55%) | P1 0.001 |
| Heterozygotes (pro/arg) | GC | 6 (30%) | 9 (22.5%) | 9 (45%) | 7 (35%) | P2 0.003 | |
| Homozygotes (pro/pro) | CC | 8 (40%) | 9 (22.5%) | 2 (5%) | 2 (10%) | P3 0.001 | |
| Frequency of alleles | G allele | 18 (45%) | 53 (66.3%) | 27 (67.5%) | 29 (72.5%) | P4 0.065 | |
| C allele | 22 (55%) | 27 (33.7%) | 13 (32.5%) | 11 (27.5%) | P5 0.416 |
P1: Compared between HCC group and LC group
P2: Compared between HCC group and CHCV group
P3: Compared between HCC group and control group
P4: Compared between LC group and CHCV group
P5: Compared between LC group and control group
| Parameters | HCC (n=20) | LC (n=40) | CHCV (n=20) | Control (n=20) | p-value |
|---|---|---|---|---|---|
| P1 | 17.286 ± 7.9 Mean ± SD |
7.19 ± 6.33 Mean ± SD |
1.519 ± 0.811 Mean ± SD |
1.2 ± 0.507 Mean ± SD |
0.0001 |
| P2 | 19 Median (Range) |
4 Median (Range) |
1.2 Median (Range) |
1.3 Median (Range) |
0.001 |
| P3 | 1.6 - 30.9 Median (Range) |
1.3 - 19.5 Median (Range) |
0.4 - 3.5 Median (Range) |
0.4 - 1.8 Median (Range) |
0.0001 |
| P4 | 0.0001 | ||||
| P5 | 0.001 |
In Table 3, we present the comparison of TP53 serum levels among the four study groups.
HCC patients exhibited a statistically significant increase in TP53 serum levels, with a mean of 17.286±7.9 (median: 19, range: 1.6-30.9), compared to LC patients, CHCV patients, and normal controls (p-value= 0.0001, 0.001, 0.0001, respectively). Additionally, LC patients showed a statistically significant increase in TP53 serum levels compared to CHCV patients and normal controls (p-value= 0.0001, 0.001, respectively).
| Groups | Genotypes | Anova | P-value | |||
|---|---|---|---|---|---|---|
| GG | GC | CC | ||||
| HCC | N | 6 | 6 | 8 | 0.036* | |
| Mean | 21.51 | 17.99 | 10.95 | |||
| SD | 5.871 | 7.56 | 7.47 | |||
| LC | N | 22 | 9 | 9 | 0.318 | |
| Mean | 4.26 | 5.81 | 7.95 | |||
| SD | 3.3 | 6.33 | 7.16 | |||
| CHCV | N | 9 | 9 | 2 | 0.834 | |
| Mean | 1.588 | 1.54 | 1.5 | |||
| SD | 0.819 | 0.919 | 0.567 | |||
| Control | N | 11 | 7 | 2 | 0.226 | |
| Mean | 1.055 | 1.29 | 1.7 | |||
| SD | 0.47 | 0.55 | 0.14 | |||
In Table 4, the relationship between plasma TP53 levels and TP53 Codon 72 genotypes in all study groups is presented.
The table illustrates the distribution of TP53 genotypes (GG, GC, CC) in the study groups, along with their respective means and standard deviations. Statistical analysis using ANOVA shows that high levels of TP53 were significantly associated with the CC genotype in the HCC group (p-value=0.036).
In this study, we investigated the role of the TP53 gene and its codon 72 polymorphism in the development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related liver cirrhosis. Our findings revealed several significant associations and shed light on potential predictive factors for HCC development.
Firstly, we observed a marked increase in TP53 serum levels in HCC patients compared to patients with liver cirrhosis related to HCV and healthy controls. This elevation in TP53 serum levels in HCC patients suggests a potential role for TP53 in the pathogenesis of HCC. It is well-established that TP53 serves as a tumor suppressor gene, and its activation can induce cell cycle arrest or apoptosis, thereby preventing carcinogenesis. However, mutations or alterations in TP53 can lead to dysfunctional TP53 protein, potentially contributing to the progression of cancer. Our results indicate that elevated TP53 levels may reflect the presence of HCC and the associated dysregulation of TP53 function.
Furthermore, we examined the distribution of TP53 codon 72 genotypes (GG, GC, CC) among the study groups. Notably, we found a statistically significant association between the CC genotype and HCC development. The CC genotype, corresponding to the proline (Pro) allele, was more frequently detected in HCC patients compared to other groups. This finding aligns with previous studies suggesting that the TP53 CC genotype may confer an increased risk for certain types of cancer, including HCC. The Proline (Pro) residue produced by the CC genotype may affect TP53's ability to induce apoptosis and regulate cell cycle progression, potentially contributing to carcinogenesis.
Our results also revealed differences in the distribution of TP53 alleles (G and C) among the study groups. The C allele was more common in the HCC group, whereas the G allele was more prevalent in the control group. This allelic frequency difference further supports the notion that the TP53 codon 72 polymorphism plays a role in HCC development.
In the multivariate binary logistic regression analysis, we identified independent predictors for the development of HCC in patients with HCV-related liver cirrhosis. Age, carriage of the TP53 CC genotype, and TP53 serum levels emerged as significant factors associated with HCC. These findings suggest that the combination of age, TP53 codon 72 genotype, and TP53 serum levels may serve as valuable predictive markers for HCC risk in this patient population.
In conclusion, our study provides valuable insights into the role of the TP53 gene and its codon 72 polymorphism in the development of hepatocellular carcinoma in patients with HCV-related liver cirrhosis. Elevated TP53 serum levels and the presence of the TP53 CC genotype were significantly associated with HCC, highlighting their potential as predictive markers for HCC risk. These findings may have clinical implications for the early detection and management of HCC in high-risk individuals.
Further research is warranted to validate these findings in larger cohorts and to explore the underlying mechanisms by which TP53 contributes to HCC development. Understanding the genetic and molecular factors driving HCC progression can aid in the development of targeted therapies and more effective strategies for HCC prevention and management in patients with HCV-related liver cirrhosis.
The Role of TP53 in the Development of Hepatocellular Carcinoma in HCV-Infected Patients. (2024, Jan 18). Retrieved from https://studymoose.com/document/the-role-of-tp53-in-the-development-of-hepatocellular-carcinoma-in-hcv-infected-patients
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