• Fisheries and Aquatic Sciences
• /
• v.19 no.4
• /
• pp.21.1-21.11
• /
• 2016

Background: The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR)-based expression study. In order to select the stable reference genes in abalone Haliotis discus hannai tissues (gill and hepatopancreas) under heavy metal exposure conditions (Cu, Zn, and Cd), 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ${\Delta}CT$ method). Results: Expression stability in the gill subset was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7 > RPL3 > RPL8 > ACTB > RPL4 > EF1A > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7) as stable references, whereas traditional housekeepers such as ${\beta}-tubulin$ (B-TU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts. Conclusions: The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiologies in this abalone species.

• Microbiology and Biotechnology Letters
• /
• v.44 no.1
• /
• pp.1-8
• /
• 2016

The purpose of this study was to assess the whitening effects of an extract from Smilax china L., which is a vine shrub belonging to the lily family. With regard to the whitening effects, 70% ethanol and water extracts from Smilax china L. showed more than 77.6% and 40.2% tyrosinase inhibition at a concentration of $1,000{\mu}l$. Furthermore, the 70% ethanol extract showed cytotoxicity of 89% at a concentration of $100{\mu}g/ml$ in melanoma cells. Western blot showed that the inhibitory effect of the 70% ethanol extract on MITF, TRP-1, TRP-2, and tyrosinase protein expression decreased by 89.9%, 46.2%, 57.6%, and 55.8%, respectively, at a concentration of $50{\mu}g/ml$. Moreover, reverse transcription-PCR showed that the inhibitory effect of the 70% ethanol extract on MITF, TRP-1, TRP-2, and tyrosinase mRNA expression decreased by 78.5%, 58.0%, 78.8%, and 70.8%, respectively, at the same concentration of $50{\mu}g/ml$ concentration. Further, realtime PCR showed that the 70% ethanol extract-induced decrease in MITF, TRP-1, TRP-2, and tyrosinase quantitative mRNA expression rate was concentration-dependent. The findings suggest that the extract from Smilax china L. has great potential as a cosmetic ingredient with whitening effects.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.4
• /
• pp.1675-1679
• /
• 2014

Objectives: MicroRNAs (miRNAs) are important regulators of many physiological and pathological processes, including tumorigenesis and metastasis. In this study, we sought to determine the underlying molecular mechanisms of metastatic cervical carcinoma by performing miRNA profiling. Methods: Tissue samples were collected from ten cervical squamous cancer patients who underwent hysterectomy and pelvic lymph node (PLN) dissection in our hospital, including four PLN-positive (metastatic) cases and six PLN-negative (non-metastatic) cases. A miRNA microarray platform with 1223 probes was used to determine the miRNA expression profiles of these two tissue types and case groups. MiRNAs having at least 4-fold differential expression between PLN-positive and PLN-negative cervical cancer tissues were bioinformatically analyzed for target gene prediction. MiRNAs with tumor-associated target genes were validated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: Thirty-nine miRNAs were differentially expressed (>4-fold) between the PLN-positive and PLN-negative groups, of which, 22 were up-regulated and 17 were down-regulated. Sixty-nine percent of the miRNAs (27/39) had tumor-associated target genes, and the expression levels of six of those (miR-126, miR-96, miR-144, miR-657, miR-490-5p, and miR-323-3p) were confirmed by quantitative (q)RT-PCR. Conclusions: Six MiRNAs with predicted tumor-associated target genes encoding proteins that are known to be involved in cell adhesion, cytoskeletal remodeling, cell proliferation, cell migration, and apoptosis were identified. These findings suggest that a panel of miRNAs may regulate multiple and various steps of the metastasis cascade by targeting metastasis-associated genes. Since these six miRNAs are predicted to target tumor-associated genes, it is likely that they contribute to the metastatic potential of cervical cancer and may aid in prognosis or molecular therapy.

• Journal of Genetic Medicine
• /
• v.19 no.2
• /
• pp.63-75
• /
• 2022

Purpose: Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility. In this study, we aimed to identify novel genes for susceptibility to osteoporosis and/or bone density. Materials and Methods: To identify differentially expressed genes (DEGs) between control and osteoporosis-induced cells, annealing control primer-based differential display reverse-transcription polymerase chain reaction (RT-PCR) was carried out in pre-osteoblast MC3T3-E1 cells. Expression levels of the identified DEGs were evaluated by quantitative RT-PCR. Association studies for the quantitative bone density analysis and osteoporosis case-control analysis of single nucleotide polymorphism (SNPs) were performed in Korean women (3,570 subjects) from the Korean Association REsource (KARE) study cohort. Results: Comparison analysis of expression levels of the identified DEGs by quantitative RT-PCR found seven genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib, and Scara5, that showed significantly different expression between the dexamethason-treated and untreated MC3T3-E1 cells and between the ovariectomized osteoporosis-induced mice and sham mice. Association studies revealed that there was a significant association between the SNPs in the five genes, ANXA6, COL5A1, ENO1, MYOF, and SCARA5, and bone density and/or osteoporosis. Conclusion: Using a whole-genome comparative expression analysis, gene expression evaluation analysis, and association analysis, we found five genes that were significantly associated with bone density and/or osteoporosis. Notably, the association P-values of the SNPs in the ANXA6 and COL5A1 genes were below the Bonferroni-corrected significance level.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.16
• /
• pp.7031-7038
• /
• 2015

Background: Arginine may play important roles in tumor progression by providing ornithine for polyamine biosynthesis, required for cell growth. The aim of this work was to determine the expression of arginine metabolic pathway enzymes in head and neck squamous cell carcinoma (HNSCC) in northeast India. Materials and Methods: The expressions of arginase isoforms (ARG1 and ARG2), ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) were examined in fifty paired HNSCC and adjacent non-tumor tissues by immunohistochemistry. Immunocytochemistry, semiquantitative reverse transcription sq-PCR and quantitative real-time qPCR were used to assess protein and mRNA expressions in peripheral blood of fifty HNSCC patients and hundred controls. Results: ARG1 and ODC protein and mRNA were strongly expressed in peripheral blood from HNSCC patients. No ARG2 expression was observed. In vivo, expression of ARG1, ARG2 and ODC was significantly higher in tumor than in non-tumor tissues. Most tumors expressed low levels of OAT, with no difference in tissues or blood, compared to controls. The absolute extent of maximal ARG1 upregulation with qPCR showed 6.23 fold increase in HNSCC. Conclusions: These findings strongly suggest that in HNSCCs, the ARG1 pathway is stimulated leading to the formation of polyamines as indicated by higher ODC expression, which promote tumor growth.

• Molecules and Cells
• /
• v.25 no.2
• /
• pp.258-264
• /
• 2008

Deschampsia antarctica is the only monocot that thrives in the tough conditions of the Antarctic region. It is an invaluable resource for the identification of genes associated with tolerance to various environmental pressures. In order to identify genes that are differentially regulated between greenhouse-grown and Antarctic field-grown plants, we initiated a detailed gene expression analysis. Antarctic plants were collected and greenhouse plants served as controls. Two different cDNA libraries were constructed with these plants. A total of 2,112 cDNA clones was sequenced and grouped into 1,199 unigene clusters consisting of 243 consensus and 956 singleton sequences. Using similarity searches against several public databases, we constructed a functional classification of the ESTs into categories such as genes related to responses to stimuli, as well as photosynthesis and metabolism. Real-time PCR analysis of various stress responsive genes revealed different patterns of regulation in the different environments, suggesting that these genes are involved in responses to specific environmental factors.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.24
• /
• pp.10585-10589
• /
• 2015

Breast cancer susceptibility gene 1 (BRCA1), mapped on chromosome 17q21, is implicated in the mechanisms of cellular DNA repair. Inactivation of this gene is involved in the development of many human cancers, including breast cancer. This study aimed to investigate the prognostic value of BRCA1 promoter hypermethylation and expression in breast cancer cases. Sixty-one breast cancers were examined for BRCA1 hypermethylation by methylation-specific polymerase chain reaction (PCR), and 45 paired normal breast tissues were analyzed for altered BRCA1 mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Aberrant methylation status in BRCA1 was detected in 15 of 61 cases (24.6%), while reduced expression was found in 7 of 45 (15.6%). BRCA1 hypermethylation was statistically associated with tumor grade III (p=0.04), a high frequency of stage IIB (p=0.02), and triple-negative phenotype (OR= 3.64, 95%CI =1.1-12.3, p=0.03). Our findings indicated that BRCA1 promoter hypermethylation is a useful prognostic marker for breast cancer.

• Journal of Marine Life Science
• /
• v.3 no.1
• /
• pp.1-8
• /
• 2018

Myosin is considered as the vital motor protein in vertebrates and invertebrates. Our present study was conducted to decipher the occurrence of myosin in dog fish (Squalus mitsukurii). We isolated one clone containing 979 bp cDNA sequence, which consisted of a complete coding sequence of 453 bp and a deduced amino acid sequence of 150 amino acids from the open reading frame with molecular weight, isoelectric point and aliphatic index are 16.72 Kda, 4.49 and 78.00, respectively. It contained 428 bp long 3' UTR with single potential polyadenylation signals (AATAAA). The predicted EF CA²⁺ binding domains were identified in residue 6-41, 83-118 and 133-150. A BLAST search indicates this protein exhibits a strong similarity to whale shark (Rhincodon typus) MLC3 (91% identical) and also house mouse (Mus musculus) MLC isoform 3f (81% identical). Phylogenetic analysis revealed that this protein is a MLC 3 isoform like protein. This protein also demonstrates highly conserved region with other myosin proteins. Homology modeling of S. mitsukuri was performed using crystal structure of Gallus gallus skeletal muscle myosin II based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR results exhibits dogfish myosin protein is highly expressed in muscle tissue.

• Journal of Marine Life Science
• /
• v.7 no.2
• /
• pp.145-153
• /
• 2022

Mercury (Hg) is a major concern in marine environment because of their bioaccumulation and biomagnification properties, and adverse effects to aquatic organisms at even a trace amount. However, little information on the effects of Hg, compared to other heavy metals, is available in marine small crustaceans. Here, we investigated the transcriptional modulation of metabolism-related genes in the brackish water flea, Diaphanosoma celebensis after exposure to sublethal concentration (0.2, 0.4, 0.8 ㎍/l) of HgCl₂ for 48 h. Relative mRNA expression levels of five detoxification enzyme-coding genes (cytochrome P450; cyp360A1, cyp361A1, cyp4AP3, cyp4C122, and cyp370C5) and six digestive enzyme-coding genes [alpha amylase (AMY), alpha amylase related protein (AMY-like), trypsin (TRYP), chymotrypsin-like protein (CHY), lipase (LIP), pancreatic lipase-related protein (PLRP)] were analyzed using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). As results, Hg increased the mRNA level of cyp370C5 (clan2) and cyp4AP3 (clan4) in a concentration dependent manner. A significant increase in TRYP mRNA was also concentration-dependently observed after exposure to Hg. These findings suggest that cyp370C5 and cyp4AP3 play a key role in Hg detoxification in D. celebensis, and Hg can affect energy metabolism by modulating the transcription of digestive enzyme. This study will provide better understanding the molecular effects of Hg in marine small crustacean.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.14
• /
• pp.5815-5818
• /
• 2014

For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously upregulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ${\beta}$-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.