The Primary Research Of VASN Protein As Serum Biomarkers And Therapeutic Targets In Liver Cancer

Liver cancer is a common malignancy, it is reported that new patients with hepatocellular carcinoma (HCC) have increased more than700,000each year in the worldwide. China is the country with the highest incidence of liver cancer in the global. The statistics show that the number of liver cancer in China accounted for55percent of the world, the number of deaths in China accounted for45percent of the world which rank the second of cancer deaths with the increasing incidence. Early diagnosis and treatments are essential to HCC patients for improvement of the prognosis and survival time. Alpha-fetoprotein (AFP) is the first discovered tumor biomarker for diagnosis, which have played an important role in cancer screening, early diagnosis, and evaluation of treatment and prognosis. However, AFP has poor sensitivity for the detection of some HCC patients, because about30percent to40percent of patients afflicted with HCC have normal levels of serum AFP, and the number is increasing these years. These patients are lack of effective tumor marker for assisted diagnosis, prognosis, and efficacy evaluation. Clearly, screening for new tumor biomarkers to improve HCC diagnosis and help HCC prognosis evaluation are urgently needed. Our research work mainly involves the discovery of new biomarker of hepatocellular carcinoma by developed SELEX and identification and potentials in HCC.Part One:The discovery and identification of HCC serum biomarkerOur research group has gained an enriched single stranded DNA pool that specifically recognized serum components of hepatic carcinoma after five rounds of positive and subtractive selections through a developed subtractive EMSA-SELEX using the AFP negative serum of HCC patient with extra-hepatic metastasis as targets and the normal serum as counter target. One of the targets, VASN protein, of the enriched pool was identified by EMSA assay using serum and labeled pool and then characterized by mass spectrometry from the band with retarded migration. The expression of VASN in serum of HCC patients, tumor cells and tumor tissues was detected. Results showed that the level of VASN in HCC patients'serum is higher than that in normal serum and has no relationship with the level of AFP, which indicated the potential of combining use of VASN and AFP to improve the sensitivity of auxiliary diagnosis. Real-time quantitative PCR and Western blot results showed that the VASN is higher expressed in HepG2and SMMC-7721than in normal liver cell (HL-7702). VASN mRNA is generally highly expressed in cancerous tissues compared to normal tissues detected by real-time quantitative PCR using cDNA microarrays. Consistently, VASN was further verified to be specifically and highly expressed in cancerous tissues of liver with a main localization at cell surface and in the intercellular substances, and lowly expressed in benign liver tissues by VASN antibody immunostaining. These data indicates that VASN probably is an important molecular biomarker which is expressed and secreted by hepatoma carcinoma cells.Part Two:The preliminary study on VASN as a therapeutic targetBased on results of our previous work, related literature report and bioinformatics analysis, we suspected that VASN may play an important role in the development and progression of liver cancer. We therefore focused on the functions of VASN using molecular biology techniques in this part. Firstly, The knockdown of VASN by RNA interference assay in hepatoma cells lead to decreased proliferation, repressed migration, increased apoptosis and inhibited epithelial-mesenchymal transition (EMT) of the cell with elevated expression of epithelial E-cadherin and down expression of N-catenin and Slug, supporting the carcinogenesis of VASN. The proliferation of hepatoma cell was also repressed by anti-VASN antibody, further suggesting the potential therapeutic target of VASN as a membrane and secreted protein. On the other hand, we over expressed VASN protein in normal hepatic cells, and the results showed that the growth of cells was increased by MTT assay and so as the migration of cells detected by transwell experiments, further confirmed the carcinogenesis of VASN.Part Three:The preliminary investigation on mechanism of VASN overexpression in liver cancerBioinformatics analysis suggests that VASN may be regulated by miR-145, which was proved to be down regulated in many cancer cell lines. Our research focused on the regulation of VASN level by miR-145and thus the changes of cell biology. Firstly, our research found that there was a negative correlation between level of VASN mRNA and that of miR-145in liver cancer cell lines and normal cell lines, indicating that decreased expression of miR-145is one of the main causes of VASN over expression. Then we confirmed that VASN mRNA is the target of miR-145via the luciferase reporter gene assay and the real-time quantitative RT-PCR experiment and Western blot experiment. Secondly, we showed that over expression of miR-145in hepatoma cells resulted in obvious changes in cell morphology with an irregular shapes and disappeared pseudopodia apophysis, companied by decreased migration and slowdown of proliferation of the cell, which are consistent with the changes caused by VASN siRNA. The changes can be recovered by a cotransfection of miR-145inhibitor. Therefore, our research elucidate that miR-145down regulate VASN level and developed its carcinogenesis.Part Four:The primary applied research of BC15as hnRNPAl-specific aptamer in hepatocarcinomaIn a previous study, we gain a BC15aptamer that specifically differentiate breast cancerous tissues from normal ones through recognizing heterogenous nuclear ribonucleoprotein Al (hnRNP Al) by a tissue-slide based SELEX. Moreover, our group has also shown that the hnRNP Al was high expressed in a variety of cancerous tissues. The status of hnRNP Al and BC15were highlighted in this research on the diagnosis and treatment of liver cancer. We elucidated that hnRNP Al was over expressed in clinical liver cancer tissues including AFP-negative ones compared to para-cancerous normal tissues and benign tissues by histological examination with BC15aptamer, suggesting that hnRNP Al may be a valuable diagnostic marker for liver cancer especially for those with negative serum AFP level and BC15be a molecular probe due to its properties of easy synthesis and direct modification and thus a simpler procedure of in situ examination than antibody. Further experiments showed that upregulation of hnRNP Al can promote the proliferation and migration of liver cells in vitro, while knockdown of hnRNP Al by RNAi and BC15inhibited the proliferation and the migration of cancerous cells, strongly suggesting that hnRNP Al is involved in the development of liver cancer, and thus may be a potential target for biological therapy. BC15has been found to have stronger inhibition effect compared with the siRNA hnRNP Al, laying a foundation for the preferential application of aptamer than siRNA against intracellular target in nucleic acid based therapeutic area.