Advances in molecular biomarkers for gastric cancer: miRNAs as emerging novel cancer markers

Abstract

Carcinoma of the stomach is one of the most prevalent cancer types in the world. Although the incidence of gastric cancer is declining, the outcomes of gastric cancer patients remain dismal because of the lack of effective biomarkers to detect early gastric cancer. Modern biomedical research has explored many potential gastric cancer biomarker genes by utilising serum protein antigens, oncogenic genes or gene families through improving molecular biological technologies, such as microarray, RNA-Seq and the like. Recently, the small noncoding microRNAs (miRNAs) have been suggested to be critical regulators in the oncogenesis pathways and to serve as useful clinical biomarkers. This new class of biomarkers is emerging as a novel molecule for cancer diagnosis and prognosis, including gastric cancer. By translational suppression of target genes, miRNAs play a significant role in the gastric cancer cell physiology and tumour progression. There are potential implications of previously discovered gastric cancer molecular biomarkers and their expression modulations by respective miRNAs. Therefore, many miRNAs are found to play oncogenic roles or tumour-suppressing functions in human cancers. With the surprising stability of miRNAs in tissues, serum or other body fluids, miRNAs have emerged as a new type of cancer biomarker with immeasurable clinical potential.

Figure 1.

Restriction Analysis of Gene Expression (RAGE) kinase profile of TSA treated gastric cancer cells. Human gastric cancer cells (NUGC) were treated with histone deacetylase inhibitor TSA (tritrostatin A) for 24 hours. Total RNA was extracted and used for PTK RAGE analysis. In short, the PTK genes were amplified using degenerate PCR primers designed with PTK conserved motif regions. We labelled the 5′-primer with fluorescent FAM tag. The final PCR product was purified and subsequently digested with various restriction enzymes before conducting the analysis with the capillary electrophoresis sequencer (ABI 3100-avant). ROX labelled size stand and NED labelled house-keeping genes (ALDOA, GPI and LDHA) were mixed and loaded in the electrophoresis chamber. The electrophoresis result was analysed with ABI GeneScan software. Each PTK gene was identified by the respective unique cut restriction enzyme and unique fragment size following capillary electrophoresis. For example, erbB2, fyn and plk1 exhibited significant expression alterations (peaks indicated by the arrows). Among them, fyn is up-regulated and erbb2 and plk1 are down-regulated. This RAGE method allows us to quickly screen the expressed PTK genes with limited tissue samples (only one PCR reaction is needed).

Figure 2.

Expression of GISP/RegIV gene. Northern blot analysis of GISP/RegIV gene was performed using tissue blots containing total RNA isolated from various human normal tissues. GISP gene was expressed predominantly in the digestive system organs: pancreas, small intestine, stomach, colon, as well as testis tissue.

Figure 3.

Increased expression of miR-196a in human gastric cancer tissues. Human gastric cancer (STAD) miRNA expression NGS short-read level-3 data was obtained from TCGA data portal. In total, we have obtained and analysed miRNA expression profiles of 192 gastric cancer tissues and 22 adjacent-tumour normal gastric tissues using the Partek Genomics Suite (version 6.6). ANOVA statistical analysis was employed and about 33 miRNAs were found to be significantly dysregulated in the gastric cancer (more than 4-fold Fold change and P < 0.05). Among them, the miR-196a is significantly overexpressed in the tumour samples.

Figure 4.

miRNAs dysregulation influences (a) gastric cancer growth, (b) cell cycle progression and (c) metastatic processes. OncomiRs promote gastric cancer cell growth and metastasis via inhibition of tumour suppressor genes. Conversely, tumour suppressing miRs suppress gastric cancer cell growth and metastasis by inhibition of oncogenes. The underlined miRNA markers indicate that they play opposite dual functions in gastric cancer.