MicroRNA and cDNA-Microarray as Potential Targets against Abiotic Stress Response in Plants: Advances and Prospects

Abiotic stresses, such as temperature (heat and cold), salinity, and drought negatively affect plant productivity; hence, the molecular responses of abiotic stresses need to be investigated. Numerous molecular and genetic engineering studies have made substantial contributions and revealed that abiotic stresses are the key factors associated with production losses in plants. In response to abiotic stresses, altered expression patterns of miRNAs have been reported, and, as a result, cDNA-microarray and microRNA (miRNA) have been used to identify genes and their expression patterns against environmental adversities in plants. MicroRNA plays a significant role in environmental stresses, plant growth and development, and regulation of various biological and metabolic activities. MicroRNAs have been studied for over a decade to identify those susceptible to environmental stimuli, characterize expression patterns, and recognize their involvement in stress responses and tolerance. Recent findings have been reported that plants assign miRNAs as critical post-transcriptional regulators of gene expression in a sequence-specific manner to adapt to multiple abiotic stresses during their growth and developmental cycle. In this study, we reviewed the current status and described the application of cDNA-microarray and miRNA to understand the abiotic stress responses and different approaches used in plants to survive against different stresses. Despite the accessibility to suitable miRNAs, there is a lack of simple ways to identify miRNA and the application of cDNA-microarray. The elucidation of miRNA responses to abiotic stresses may lead to developing technologies for the early detection of plant environmental stressors. The miRNAs and cDNA-microarrays are powerful tools to enhance abiotic stress tolerance in plants through multiple advanced sequencing and bioinformatics techniques, including miRNA-regulated network, miRNA target prediction, miRNA identification, expression profile, features (disease or stress, biomarkers) association, tools based on machine learning algorithms, NGS, and tools specific for plants. Such technologies were established to identify miRNA and their target gene network prediction, emphasizing current achievements, impediments, and future perspectives. Furthermore, there is also a need to identify and classify new functional genes that may play a role in stress resistance, since many plant genes constitute an unexplained fraction.

Icariin and Icariside II Reciprocally Stimulate Osteogenesis and Inhibit Adipogenesis of Multipotential Stromal Cells through ERK Signaling

Herba Epimedii is a famous Chinese herbal medicine for treating bone diseases. Icariin and icariside II, the main chemical constituents, have attracted great attention from scientists for their potential as antiosteoporosis agents. Our study aimed to evaluate their effects on the lineage commitment of multipotential stromal cells (MSCs). The osteogenesis and adipogenesis of MSCs were assessed by ALP activity, calcium deposition, and adipocyte formation. The expression profiles and levels of osteogenic and adipogenic specific genes were evaluated by cDNA microarray and quantitative real-time PCR. The involvement of extracellular signal-regulated kinase (ERK) signaling was studied by enzyme-linked immunosorbent assay. Icariin and icariside II significantly increased ALP activity and mineralization during osteogenic differentiation of MSCs. Runx2, Col1, and Bmp2 were upregulated in the presence of icariin and icariside II. Meanwhile, they downregulated Pparg, Adipsin, and Cebpb expression during adipogenic differentiation. cDNA microarray revealed 57 differentially expressed genes during lineage commitment of MSCs. In addition, icariin and icariside II enhanced the phosphorylation of ERK, and the above biological effects were blocked by ERK inhibitor U0126. Icariin and icariside II may drive the final lineage commitment of MSCs towards osteogenesis and inhibit adipogenesis through the ERK signaling pathway. Both of them exert multiple osteoprotective effects and deserve more attention for their medicinal and healthcare prospects.

The Effects and Underlying Mechanisms of Hepatitis B Virus X Gene Mutants on the Development of Hepatocellular Carcinoma

Background: We aimed to elucidate the mechanism by which hepatitis B virus X (HBx) gene mutations increase the risk of hepatocellular carcinoma (HCC) and identify novel therapeutic targets.Methods: Wild-type and four HBx mutants (M1, A1762T/G1764A; M2, T1674G+T1753C+A1762T/G1764A; M3, C1653T+T1674G+A1762T/G1764A; Ct-HBx, carboxylic acid-terminal truncated HBx) were delivered into the livers of fumarylacetoacetate hydrolase-deficient mice by using the Sleeping Beauty (SB) transposon system, respectively. Seven liver tissues and seven tumor tissues of the SB mouse models were subjected to HBV-capture sequencing. Three liver tissues from WT-HBx mice, three tumor tissues from M3-HBx mice, and three tumor tissues from Ct-HBx mice were subjected to cDNA microarray analysis. HeLa cells stably expressing WT-HBx and the four HBx mutants were also subjected to cDNA microarray assay.Results: The incidence of HCC was higher in the mice injected with M3-HBx or Ct-HBx. M3-HBx had a stronger capacity of upregulating inflammatory cytokines than other HBx variants. HBV-capture sequencing showed that the HBx fragments were mainly integrated into intergenic and intron regions. No significant difference was observed in the number of insertion sites between tumors and liver tissues. Ectopic expression of the HBx mutants, especially M3-HBx and Ct-HBx, significantly increased cell proliferation and the S phase proportion of HepG2 and HeLa cells, compared to WT-HBx. Liver tissues of the SB mice and the transfected cells were subjected to cDNA microarray analysis. Plasminogen activator inhibitor-1 (PAI1) and cell division cycle 20 (CDC20) were identified as novel effectors. M3-HBx and Ct-HBx significantly upregulated the expression of PAI1 and CDC20 in HepG2 and HeLa cells as well as the livers of the SB mice. PAI1 silencing attenuated the effect of M3-HBx and Ct-HBx on the growth of HepG2 cells and greatly decreased the growth of HeLa cells with Ct-HBx. Conclusion: HBx C1653T+T1674G+A1762T/G1764A mutant and Ct-HBx promote carcinogenesis via upregulating PAI1 and CDC20. PAI1, an important player bridging the HBx mutants and HCC, should be a promising candidate as a predictive and prognostic biomarker and therapeutic target in HBV-related HCC.

The effect of 4-hexylresocinol administration on SCC-9 cells: mass spectrometric identification of proteins and cDNA microarray analysis

Background In stress situations, bacteria produce dormancy-inducing factors to stop cell growth. The dormancy-inducing factors may have an inhibitory effect on tumor cell growth. Here we analyzed the differentially expressed protein profiles after 4-hexylresorcinol (4HR), one of the dormancy-inducing factors, administration using in vitro oral squamous carcinoma cells (SCC-9). Method The control group was SCC-9 cells culture without 4HR administration. The experimental group received 10 μg/mL of 4HR. Collected proteins from each group were loaded for 2D electrophoresis. Among the separated proteins, 20 differentially expressed proteins were selected and processed for LC-MS/MS. Results In proteomic analysis, the expression of keratin 1, keratin 10, and histone H2B were increased. In cDNA microarray assay, the genes related to the cellular differentiation (involucrin, keratin 13, 14) were highly expressed in the 4HR treated group (fold ratio > 2.0; Table 2). Interestingly, histone family was upregulated in the cDNA microarray assay. Conclusion The administration of 4HR on SCC-9 cells increased epithelial cell differentiation markers and histone.

Acrolein Contributes to Human Colorectal Tumorigenesis Through Activation of RAS/MAPK Pathway

Background. Colorectal cancer (CRC) is one of the most well-known malignancies with high prevalence and poor 5-year survival. Previous studies have demonstrated that intake of food rich in fat, and with low fiber content (as known as high-fat diet, HFD) is capable of increasing the odds of developing CRC. Acrolein, an IARC group 2A carcinogen, can be formed by thermal treatment of animal and vegetable fats, carbohydrates and amino acids through Maillard reaction. Also, acrolein has been shown to be produced from microbial glycerol metabolism in human gut. Consequently, humans are at risk of acrolein exposure through consumption of foods rich in fat. However, whether acrolein contributes to HFD-induced CRC tumorigenesis remains elusive.Methods. The effect of acrolein in oncogenic transformation was analyzed using NIH/3T3 cells with xenograft tumorigenesis mice models. Furthermore, cDNA microarray analysis with Ingenuity Pathway Analysis (IPA) was performed in acrolein-transformed NIH/3T3 cells. Finally, acrolein-induced DNA damages (Acr-dG) were analyzed in tumor tissues and normal epithelial of CRC patients using immunohistochemical analysis. The levels of Acr-dG adducts were associated with tumor characteristics and CRC patients’ survival using Chi-Square analysis and Kaplan Meier survival analysis, respectively.Results. In this present study, we found that acrolein induced oncogenic transformation including faster cell cycling, proliferation, soft agar formation, sphere formation, cell migration in NIH/3T3 cells. Using xenograft tumorigenicity assays, the acrolein-transformed NIH3T3 clone formed tumors whereas no tumors were observed in mice inoculated with NIH3T3 parental cells. In addition, RAS/MAPK pathway contributing to colon tumorigenesis was activated in NIH/3T3 Acr-clone using cDNA microarray analysis with IPA. Finally, Acr-dG adducts were higher in CRC tumor tissues compared to normal epithelial cells in CRC patients. Intriguingly, CRC patients with higher Acr-dG adducts have better prognosis. Conclusions. Taken together, this is the first study to demonstrate that acrolein is important in oncogenic transformation through activating RAS/MAPK signaling pathway contributing to colon tumorigenesis. Thus, acrolein might be a novel target for early detection, prevention and treatment of tumors in the future.

Hepatitis B Virus preS/S Truncation Mutant rtM204I/sW196* Increases Carcinogenesis through Deregulated HIF1A, MGST2, and TGFbi

Inevitable long-term therapy with nucleos(t)ide analogs in patients with chronic hepatitis B virus (HBV) infection has selected reverse-transcriptase (rt) mutants in a substantial proportion of patients. Some of these mutants introduce premature stop codons in the overlapping surface (s) gene, including rtA181T/sW172*, which has been shown to enhance oncogenicity. The oncogenicity of another drug-resistant mutant, rtM204I/sW196*, has not been studied. We constructed plasmids harboring rtM204I/sW196* and assessed the in vitro cell transformation, endoplasmic reticulum (ER) stress response, and xenograft tumorigenesis of the transformants. Cellular gene expression was analyzed by cDNA microarray and was validated. The rtM204I/sW196* transformants, compared with the control or wild type, showed enhanced transactivation activities for c-fos, increased cell proliferation, decreased apoptosis, more anchorage-independent growth, and enhanced tumor growth in mouse xenografts. X box-binding protein-1 (XBP1) splicing analysis showed no ER stress response. Altered gene expressions, including up-regulated MGST2 and HIF1A, and downregulated transforming growth factor beta-induced (TGFbi), were unveiled by cDNA microarray and validated by RT-qPCR. The TGFbi alteration occurred in transformants with wild type or mutated HBV. The altered MGST2 and HIF1A were found only with mutated HBV. The rtM204I/sW196* preS/S truncation may endorse the cell transformation and tumorigenesis ability via altered host gene expressions, including MGST2, HIF1A, and TGFbi. Downregulated TGFbi may be a common mechanism for oncogenicity in HBV surface truncation mutants.