Hepatocellular carcinoma (HCC) is the main histological subtype of liver cancer, accounting for about 90% of primary liver cancers, usually caused by hepatitis B/C virus It is the third most common cause of cancer-related mortality worldwide. Due to the lack of sensitive diagnostic methods and biomarkers, most HCC patients are diagnosed at an advanced stage when effective treatment is no longer available. Therefore, there is an urgent need for accurate and effective biomarkers that can diagnose HCC patients at an early stage.
tsRNA is a non-coding small RNA of about 18-40 nucleotides in length produced by tRNA (transfer RNA) under the action of nuclease RNaseZ or Dicer. Studies have shown that tsRNAs play a crucial role in the generation and development of various cancers, for example, in B-cell chronic lymphocytic leukemia (CLL), ts-3676 may function as a tumor suppressor. Notably, tsRNA is relatively stable in serum, which may be a biomarker for a variety of diseases, including pancreatic cancer, breast cancer, and head and neck squamous cell carcinoma.
Recently, Nanjing University Yanbo Wang Team, Nanjing University of Traditional Chinese Medicine Fangfang Jin Team and Lianyungang First People’s Hospital Liuqing Yang teamcooperationin Frontiers of Medicine “Serum mitochondrial tsRNA serves as a novel biomarker for hepatocarcinoma diagnosis” in . The research team identified a new tsRNA in the serum of liver cancer patients by RNA sequencingand quantitative reverse transcription PCR ( RT-qPCR )——tRF-Gln-TTG-006, can be used as a promising blood biomarker to help doctors diagnose HCC patients as early as possible. In addition, the research team also discovered that tsRNA has potential biological functions during HCC progression.
The article was published in >Frontiers of Medicine
In order to verify whether tsRNA in serum can be used as a biomarker for blood-based HCC detection, the research team used high-pass tsRNA including tsRNA Quantitative sequencing, RT-qPCR validation was performed on a single sample (Figure 1). Total RNA was extracted and sequenced from the pooled sera of 30 HCC patients and 30 age- and sex-matched healthy controls, 233 tsRNAs were screened, of which 110 showed dysregulation (59 up-regulated, 51 down-regulated) (Fig. 2A and 2B). The analysis showed that compared with healthy controls, there were 17 tsRNAs that were significantly differentially expressed in the serum of HCC patients.
Figure 1. Flow chart of study design. Source: Frontiers of Medicine
Next, the research team analyzed the serum of 24 healthy controls and 24 HCC patients by absolute quantification samples and assessed the top 5 tsRNAs in the samples that exhibited upregulation using probe-based RT-qPCR. The analysis showed that two tsRNAs (tRF-Pro-AGG-005 and tRF-Gln-TTG-006) were significantly elevated in serum of HCC patients compared with healthy controls (Fig. 2C – Figure 2G), indicating that tRF-Pro-AGG-005 and tRF-Gln-TTG-006 are the most promising tsRNAs to differentiate HCC patients from healthy individuals >.
Figure 2. Analysis of differentially expressed serum tsRNA in HCC. Source: Frontiers of Medicine
In order to identify whether the above tsRNA has diagnostic value, researchThe research team studied two large independent serum sample cohorts (80 HCC patients and 79 healthy controls from Lianyungang First People’s Hospital; 73 HCC patients and 76 healthy controls from Drum Tower Hospital Affiliated to Nanjing University School of Medicine). The expression profiles of tRF-Pro-AGG-005 and tRF-Gln-TTG-006 were further analyzed (Fig. 3A and 3B ). The results showed that Compared with the control group, the concentration of tRF-Gln-TTG-006 in the serum of HCC patients was significantly increased (P<0.0001); tRF-Pro-AGG-005 had little difference between the two groups< /strong>.
Next, the research team used ROC analysis to assess the sensitivity and specificity of risk score-based prediction, further confirming the diagnostic value of tRF-Gln-TTG-006 . The results showed that tRF-Gln-TTG-006 showed high diagnostic utility, with AUC of 0.875, sensitivity of 80.4%, and specificity of 79.4%. In addition, the research team also compared the diagnostic efficacy of tRF-Gln-TTG-006 with the clinical HCC biomarker alpha-fetoprotein (AFP). ROC analysis showed that tRF-Gln-TTG-006 had better diagnostic performance than AFP, especially in the diagnosis of early HCC (stage I) patients, the accuracy of tRF-Gln-TTG-006 was significantly higher< /strong>.
Figure 3. Diagnostic analysis of tRF-Gln-TTG-006 in two cohorts from different hospitals. Source: Frontiers of Medicine
The research team analyzed the expression levels of tsRNA in HCC tissues and paired serum of 11 HCC patients and found that tRF-Gln-TTG The expression level of -006 was positively correlated between them (Fig. 4). To further analyze whether the up-regulated tRF-Gln-TTG-006 was released from HCC cells, the research team cultured HepG2 cells (a human HCC cell line) and detected the expression of tsRNA in the culture medium of different numbers of cells. The results showed that the expression of tRF-Gln-TTG-006 was proportional to the number of cells, indicating that tRF-Gln-TTG-006 was derived from HCC cells (Figure 4) .
MINTbase alignment showed that tRF-Gln-TTG-006 matched mitochondrial tRNA and was located at positions 1-22 of the tRNA secondary structure. Therefore, the research team speculated that tRF-Gln-TTG-006 may have a similar function to miRNA. To explore the potential function of tRF-Gln-TTG-006, the research team used RNAhybrid to predict the target genes of tRF-Gln-TTG-006, 143 potential target genes were obtained in total. Gene Ontology (GO) enrichment analysis showed that thesetarget genes were enriched in and tumorstumor progressionmainly Stepsrelatedrelatedcadherin and β-catenin.
Further, the research team transferred the tRF-Gln-TTG-006 mimic into HepG2 and HuH-7 cells (Figure 4D –Fig.4F), found that tRF-Gln-TTG-006 reduced the colony of two HCC cell lines Forming ability. The apoptosis assay showed that tRF-Gln-TTG-006 induced a higher apoptosis rate in HuH-7 cells (Figure 4G and 4H). The above results indicated that tRF-Gln-TTG-006 may act as a tumor suppressor and be released by HCC cells.
Figure 4. Biological function of tRF-Gln-TTG-006. Source: Frontiers of Medicine
In this study, high-throughput RNA sequencing and multiple separate qRT-PCR assessments were performed in HCC patients. Unique serum tsRNA signatures are defined in . In particular, the research team identified a novel serum tsRNA, tRF-Gln-TTG-006, in HCC patients, which has an excellent ability to distinguish HCC patients from healthy controls and can be used as a valuable tool in the early diagnosis of HCC. Biomarkers.
References:Zhan, S., Yang, P., Zhou, S. et al. Serum mitochondrial tsRNA serves as a novel biomarker for hepatocarcinoma diagnosis. Frontiers of Medicine, 1-11 (2022). doi: 10.1007/s11684-022-0920-7Source: Sequencing China