Dataset Information

A protein-based set of reference markers for liver tissues and hepatocellular carcinoma.

ABSTRACT:

Background

During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression.

Methods

A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the identified reference markers.

Results

The expression levels of three protein markers beta-actin (ACTB), heat shock protein 60 (HSP60), and protein disulphide isomerase (PDI) were found to be stable using p-values (p > 0.99) as a ranking tool in all 224 human liver tissues examined by 2-DE analysis. Of high importance, ACTB and HSP 60 were successfully validated at both protein and mRNA levels in human hepatic tissues by western blot, immunohistochemistry and real-time quantitative PCR. In addition, no significant correlation of these markers with any clinicopathological features of HCC and cirrhosis was found. Gene stability measure of these two markers with other conventionally applied housekeeping genes was assessed by the geNorm algorithm, which ranked ACTB and HSP60 as the most stable genes among this cohort of clinical samples.

Conclusion

Our findings identified 2 reference markers that exhibited stable expression across human liver tissues with different conditions thus should be regarded as reliable reference moieties for normalisation of gene and protein expression in clinical research employing human hepatic tissues.

SUBMITTER: Sun S

PROVIDER: S-EPMC2742551 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Publications

A protein-based set of reference markers for liver tissues and hepatocellular carcinoma.

Sun Stella S Yi Xin X Poon Ronnie Tp RT Yeung Chun C Day Philip J R PJ Luk John M JM

BMC cancer 20090902

<h4>Background</h4>During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed ...[more]

PMID: 19725976

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Project description:The physiological role of autophagy in the progression of liver diseases is still debated. To understand the clinical relevance of autophagy in primary e secondary hepatic tumors, we analyzed the expression of mTOR (mammalian target of rapamycin), a key regulator of autophagy; Raptor (regulatory-associated protein of mTOR); ULK1 (Unc-51 like kinase 1) determinant in the autophagy initiation; LC3 (microtubule-associated protein 1A/1B-light chain 3), a specific marker of autophagosomes; and p62, a selective autophagy receptor. Samples from subjects with chronic hepatitis (n.58), cirrhosis (n.12), hepatocellular carcinoma (HCC, n.56), metastases (n.48) from colorectal cancer and hyperplasia or gallbladder stones (n.7), the latter considered as controls, were examined. Gene expression analysis was carried out in n.213 tissues by absolute q-PCR, while protein expression by Western Blot in n.191 lysates, including tumoral, surrounding tumoral and normal tissues. Nonparametric statistical tests were used for comparing expression levels in the above-mentioned groups. Subgroup analysis was performed considering viral infection and chemotherapy treatment. The mTOR transcriptional level was significantly lower in metastases compared to HCC (P = 0.0001). p-mTOR(Ser2448) and LC3II/LC3I protein levels were significantly higher in metastases compared to HCC (P = 0.008 and P<0.0001, respectively). ULK(Ser757) levels were significantly higher in HCC compared to metastases (P = 0.0002) while the HCV- and HBV- related HCC showed the highest p62 levels. Chemotherapy induced a down-regulation of the p-mTOR(Ser2448) in metastases and in non-tumor surrounding tissues in treated patients compared to untreated (P = 0.001 and P = 0.005, respectively). Conclusions: the different expression of proteins considered, owning their interaction and diverse tissue microenvironment, indicate an impairment of the autophagy flux in primary liver tumors that is critical for the promotion of tumorigenesis process and a coexistence of autophagy inhibition and activation mechanisms in secondary liver tumors. Differences in mTOR and LC3 transcripts emerged in tumor-free tissues, therefore particular attention should be considered in selecting the control group.

Project description:Background: It is a challenge to identify those patients who, after undergoing potentially curative treatments for hepatocellular carcinoma, are at greatest risk of recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissues. Methods: We aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival. Results: The expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (p = 0.04). Conclusions: We have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlating with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma. Keywords: Hepatocellular carcinoma, Expression array, Illumina, Signatures, Outcome prediction Training cohort: 80 tumor and 82 non-tumor liver tissues surgically resected from patients with hepatocellular carcinoma (HCC); Validation cohort: 225 non-tumor liver tissues surgically resected from patients with HCC. Clinical data has been withheld from GEO due to privacy concerns.

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Dataset Information

A protein-based set of reference markers for liver tissues and hepatocellular carcinoma.

Background

Methods

Results

Conclusion

Publications

A protein-based set of reference markers for liver tissues and hepatocellular carcinoma.

Similar Datasets

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