Comparative de novo transcriptome analysis of flower and root of Oliveria decumbens Vent. to identify putative genes in terpenes biosynthesis pathway.

The Oliveria decumbens Vent. is a wild, rare, annual medicinal plant and endemic plant of Iran that has metabolites (mostly terpenes) which make it a precious plant in Persian Traditional Medicine and also a potential chemotherapeutic agent. The lack of genetic resources has slowed the discovery of genes involved in the terpenes biosynthesis pathway. It is a wild relative of Daucus carota. In this research, we performed the transcriptomic differences between two samples, flower and root of Oliveria decumbens, and also analyze the expression value of the genes involved in terpenoid biosynthesis by RNA-seq and its essential oil's phytochemicals analyzed by GC/MS. In total, 136,031,188 reads from two samples of flower and root have been produced. The result shows that the MEP pathway is mostly active in the flower and the MVA in the root. Three genes of GPP, FPPS, and GGPP that are the precursors in the synthesis of mono, di, and triterpenes are upregulated in root and 23 key genes were identified that are involved in the biosynthesis of terpenes. Three genes had the highest upregulation in the root including, and on the other hand, another three genes had the expression only in the flower. Meanwhile, 191 and 185 upregulated genes in the flower and root of the plant, respectively, were selected for the gene ontology analysis and reconstruction of co-expression networks. The current research is the first of its kind on Oliveria decumbens transcriptome and discussed 67 genes that have been deposited into the NCBI database. Collectively, the information obtained in this study unveils the new insights into characterizing the genetic blueprint of Oliveria decumbens Vent. which paved the way for medical/plant biotechnology and the pharmaceutical industry in the future.

The combined restoration of miR-424-5p and miR-142-3p effectively inhibits MCF-7 breast cancer cell line via modulating apoptosis, proliferation, colony formation, cell cycle and autophagy.

BACKGROUND: The combined restoration of tumor-suppressive microRNAs (miRs) has been identified as a promising approach for inhibiting breast cancer development. This study investigated the effect of the combined restoration of miR-424-5p and miR-142-3p on MCF-7 cells and compared the efficacy of the combined therapy with the monotherapies with miR-424-5p and miR-142-3p. METHODS: After transfection of miR-424-5p and miR-142-3p mimics into MCF-7 cells in the combined and separated manner, the proliferation of tumoral cells was assessed by the MTT assay. Also, the apoptosis, autophagy, and cell cycle of the cells were analyzed by flow cytometry. Western blot and qRT-PCR were used to study the expression levels of c-Myc, Bcl-2, Bax, STAT-3, Oct-3, and Beclin-1. RESULTS: Our results have demonstrated that the combined restoration of miR-424-5p and miR-142-3p is more effective in inhibiting tumor proliferation via upregulating Bax and Beclin-1 and downregulating Bcl-2 and c-Myc. Besides, the combined therapy has arrested the cell cycle in the sub-G1 and G2 phases and has suppressed the clonogenicity via downregulating STAT-3 and Oct-3, respectively. CONCLUSION: The combined restoration of miR-424-5p and miR-142-3p is more effective in inhibiting MCF-7 breast cancer development than monotherapies with miR-424-5p and miR-142-3p.

MiRNA-138-5p: A strong tumor suppressor targeting PD-L-1 inhibits proliferation and motility of breast cancer cells and induces apoptosis.

MicroRNAs are important regulators in multiple cellular processes and are closely related to a variety of cancers including breast cancer (BC). Immunotherapy using different methods such as modulating immune check points has been known as an advanced and successful procedure in cancer treatment. Here we investigated the effects of miRNA-138-5p restoring on Programmed Death Ligand 1 (PD-L-1) expression, BC biological behaviors and T-cell exhaustion. Breast cancer specimens and cell lines were provided and qRT-PCR and western blotting were used to measure the expression of miRNA-138-5p, PD-L-1 and other underlying genes. MTT and colony formation assays and scratch test were employed to specify proliferation, cloning and migration in miRNA-138-5p-transfected MDA-MB-231 cells respectively. DAPI staining assay and flow-cytometry were used to investigate apoptosis rate and cell cycle development. Finally, isolated T-cells were co-cultured with transfected BC cells to explore the effect of miRNA-138-5p on T-cell exhaustion. qRT-PCR revealed down-regulation ofmiRNA-138-5p conversely, up-regulation of PD-L-1 in BC tissues and cell lines. Transfection of miRNA-138-5p into MDA-MB-231 cells inhibited PD-L-1 expression. Western blotting, MTT and colony formation assays affirmed the anti-proliferative effect ofmiRNA-138-5p through down-regulating PI3K/AKT pathway. Also, miRNA-138-5p induced apoptosis in BC cells via up-regulating Caspase-9 and Caspase-3 and arresting cell cycle at sub-G1 phase. Moreover, scratch test and western blotting indicated that miRNA-138-5p inhibits cell motility via targeting MMP2, MMP9 and vimentin but up-regulating E-cadherin. Finally, miRNA-138-5p restrains T-cell exhaustion via suppressing PD-L-1 expression in BC cells leading to disrupt PD-L-1/PD-1 interaction and modulate effector cytokines in T-cells.

MicroRNA-424-5p enhances chemosensitivity of breast cancer cells to Taxol and regulates cell cycle, apoptosis, and proliferation.

Combination therapy has been considered as a potential method to overcome the BC chemoresistance. MicroRNAs (miRs) have been suggested as a therapeutic factor in the combination therapy of BC. This project aimed at examining the possible activity and molecular function of miR-424-5p and Taxol combination in the human BC cell line. MDA-MB-231 cells were treated with miR-424-5p mimics and Taxol, in a combined manner or separately. We used the MTT test for assessing the cell proliferation. In addition, flow-cytometry was used for evaluating apoptosis and cell-cycle. Expression levels of underlying molecular factors of miR-424-5p were assessed using western-blotting and qRT-PCR. The obtained results demonstrated that miR-424-5p repressed BC cell proliferation and sensitized these cells to Taxol treatment through the induction of apoptosis. Further investigations showed that miR-424-5p might increase BC chemosensitivity through the regulation of apoptosis-related factors including P53, Caspase-3, Bcl-2, and Bax as well as the proliferation-related gene c-Myc. Moreover, miR-424-5p restoration in combination with Taxol treatment decreased the colony formation by regulating Oct-4 and led to G2 arrest via modulating Cdk-2 expression. Western-blotting demonstrated that miR-424-5p may perform its anti-chemoresistance role by regulating the PD-L1 expression and controlling PTEN/PI3K/AKT/mTOR. Overall, the upregulation of miR-424-5p was indicated to upregulate the sensitivity of BC cells to treatment with Taxol. MiR-424-5p might regulate the chemosensitivity of the BC cell line by modulating PD-L1 and controlling the PTEN/mTOR axis. Therefore, the combination of miR-424-5p with Taxol would represent a novel procedure to treat against BC.

BC032913 as a Novel Antisense Non-coding RNA is Downregulated in Gastric Cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) are a subset of non-coding RNAs. Abnormal expression of lncRNAs is associated with the development of cancers including gastric cancer (GC). Increasing evidence in this field reveals that lncRNAs have a key role in biological actions via modulating the expression of genes at various genomic levels. BC032913, as a novel antisense lncRNA, may have a role in GC progression. OBJECTIVE: The very aim of this study was to examine the clinical significance and the functional role of lncRNA BC032913 in GC. METHODS: QRT-PCR technique was exerted to survey the expression level of BC032913 in GC tissues and matched adjacent non-tumorous tissues. Furthermore, the association between clinicopathological parameters and the expression level of BC032913 was evaluated. RESULTS: BC032913 expression was downregulated in cancerous tissues as compared with adjacent non-tumorous tissues (P = 0.0108). Downregulation of BC032913 expression did not demonstrate a significant association with clinicopathological parameters. CONCLUSION: LncRNA BC032913 may be considered as an impressive therapeutic target for GC.

Study of long non-coding RNA highly upregulated in liver cancer (HULC) in breast cancer: A clinical & in vitro investigation.

Background & objectives: Breast cancer remains the most common malignancy among women worldwide. Long non-coding RNAs (lncRNAs) have been shown to play critical roles in tumour initiation and progression. This study was aimed to evaluate the potential role of lncRNA highly upregulated in liver cancer (HULC) in breast cancer. Methods: The expression of HULC was evaluated in breast cancer patients and cell lines using real-time quantitative reverse transcription polymerase chain reaction. Small interfering RNA-based knockdown was also employed to study the potential role of HULC in breast cancer cell lines including ZR-75-1, MCF7 and MDA-MB-231. Results: HULC was significantly upregulated in tumour tissues compared to non-tumoural margins (P <0.001). The receiver operating characteristic (ROC) curve analysis demonstrated the biomarker potential of HULC (ROCAUC=0.78, P <0.001). The HULC knockdown induced apoptosis and suppressed cellular migration in breast cancer cell lines. Interpretation & conclusions: Our results indicated that HULC was upregulated in breast cancer and might play a role in tumourigenesis. The HULC may have a potential to be exploited as a new biomarker and therapeutic target in breast cancer.

Anti-Cancer Effect of Melatonin via Downregulation of Delta-like Ligand 4 in Estrogen-Responsive Breast Cancer Cells.

BACKGROUND: The Notch signaling pathway has a key role in angiogenesis and Delta - Like Ligand 4 (DLL4) is one of the main ligands of Notch involved in cell proliferation in sprouting vessels. OBJECTIVE: In this study, we aimed to evaluate the expression of DLL4 in primary breast tumors and to examine the effect of melatonin on DLL4 expression in vitro. METHODS: Eighty-five breast tumor and paired adjacent non-tumor tissue samples were collected. Apoptosis assay was performed on breast cancer cells to evaluate melatonin effects. Western blot and quantitative RT-PCR were used to measure DLL4 expression. Then, we investigated the effect of melatonin on the expression of DLL4 in four breast cancer cell lines at RNA and protein levels. We also performed a probabilistic neural network analysis to study genes closely associated with DLL4 expression. RESULTS: Our results showed a significantly higher expression of DLL4 in tumor tissues compared to non-tumor tissues (P = 0.027). Melatonin treatment substantially attenuated DLL4 expression in BT474 and MCF-7 cells, but not in SK-BR-3 and MDA-MB-231 cells. Also, melatonin induced apoptosis in all four cell lines. Network analysis revealed a set of 15 genes that had close association and interaction with DLL4. DLL4 was overexpressed in breast cancer tissues as compared to the non-tumor tissues. CONCLUSION: It can be concluded that melatonin treatment attenuated DLL4 expression only in estrogen- responsive breast cancer cells and is able to induce apoptosis in breast cancer cells.

Tumor suppressive activity of miR-424-5p in breast cancer cells through targeting PD-L1 and modulating PTEN/PI3K/AKT/mTOR signaling pathway.

AIMS: MicroRNAs (miRs) are key modulators of cellular processes such as proliferation, apoptosis, as well as anti-cancer immune responses. Here, we evaluated the role of miR-424-5p in breast cancer (BC) and investigated its effects on T cell-related immune response. MAIN METHODS: BC tissues and cell lines were prepared and the expression of miR-424-5p and PD-L1, as well as the underlying molecular pathways, were assessed via qRT-PCR and western blotting. The MTT assay and flow cytometry were used to assess the effect of miR-424-5p on proliferation, apoptosis, autophagy, and cell cycle progression. The co-culture of T cells with MDA-MB-231 was performed for evaluating the role of miR-424-5p in rescuing T cell exhaustion. KEY FINDINGS: The results indicated the down-regulation of miR-424-5p and up-regulation of PD-L1 expression in BC tissue specimens. MiR-424-5p transfection into PD-L1 overexpressing MDA-MB-231 cells decreased the expression of PD-L1. Also, miR-424-5p could reduce MDA-MB-231 cell viability through modulating apoptosis and autophagy pathways. Furthermore, miR-424-5p transfection leads to decreased colony formation and increased cell number at the G2/M phase. Western blot analysis illustrated that miR-424-5p could exert its anti-proliferative effect via modulating PTEN/PI3K/AKT/mTOR pathway. Moreover, it was demonstrated that suppression of PD-L1 by miR-424-5p could participate in regulating the expression of effector cytokines in T cells. SIGNIFICANCE: MiR-424-5p could be considered as a potential tumor-suppressor miR in regulating BC cellular growth, apoptosis, and T cell-related immune response through targeting PD-L1, and its downstream mediators. Therefore, we recognized miR-424-5p as a promising candidate for miR restoration therapy in BC patients.

Contribution of DNA methylation and EZH2 in SRBC down-regulation in gastric cancer.

Gastric cancer (GC), a high mortality malignancy, is induced by genetic and epigenetic factors. DNA and histone methylation play critical roles in tumor suppressor genes inactivation. SRBC (serum deprivation response factor-related gene product that binds to the c-kinase), suggested as a tumor suppressor gene, participates in apoptosis, tumor chemoresistance and DNA damage response and is repressed in various cancers. Inspecting the mechanisms underlying SRBC suppression is important for cancer treatments. We investigated SRBC promoter DNA methylation status and expression of SRBC and EZH2 histone methyltrasferase in gastric cancer. Also, we surveyed SRBC expression after 5-azacitidine and UNC1999 treatments of AGS cell line. In current work, we used gastric adenocarcinoma tissues, marginal samples and normal gastric biopsies. DNA methylation was detected by Methylation- Specific PCR and mRNA expression was measured by Real-Time PCR. SRBC promoter methylation analysis, showed fully and partial methylated versions that were associated with patient's age (p = 0.001). SRBC expression significantly decreased in GC compare with marginal and normal samples (p-value < 0.001). EZH2 showed remarkable up-regulation in GC than controls and demonstrated a strong inverse correlation with SRBC expression (r = - 0.69). Restoration of SRBC expression was observed after 5-azacitidine and UNC1999 applications with a remarkable increase by combinational treatment. We showed that EZH2 plays role in SRBC silencing in addition to DNA methylation. Our study, suggests that DNA methylation and EZH2 are involved in SRBC silencing and their inhibitors can be considered in cancer treatment investigations to overcome chemoresistance induced by SRBC inactivation.

Clinical and in vitro Study of Novel Long Non-Coding RNA lncUSMycN in Breast Cancer

Background: Despite recent advances in diagnosis and treatment, breast cancer remains a leading cause of death in women worldwide. Long non-coding RNAs are a new class of RNA molecules that have been shown to participate in tumorigenesis. The aim of this study was to investigate the expression of lncUSMycN in tumor samples and to evaluate its potential role in the breast cancer cell line. Methods: Real-time polymerase chain reaction was employed to assess lncUSMycN expression in breast tumor tissues and cancer cell lines. Furthermore, small interfering RNA was used to knockdown lncUSMycN. Results: The data showed the significant up-regulation of lncUSMycN in tumor tissues compared to non-tumor specimens (95% CI, p = 0.002). Receiver operating characteristic (ROC) curve analysis demonstrated the biomarker potential of lncUSMycN (ROCAUC = 0.70, p < 0.001) for invasive breast ductal carcinoma. Furthermore, lncUSMycN knockdown induced apoptosis and suppressed cellular migration in breast cancer cells (p < 0.01). Conclusion: The findings highlight the pivotal role of lncUSMycN in tumorigenesis, providing a new potential target for breast cancer therapy.

Boswellia serrata gum resin aqueous extract upregulatesBDNF but not CREB expression in adult male rat hippocampus.

BACKGROUND/AIM: Boswellia from the family Burseraceae has been proposed for prevention of amnesia; however, the molecular mechanism by which it affects memory is not clear. To reveal the potential molecular mechanism, the effects of boswellia on the expression of two memory related genes, CREB and BDNF, were investigated. MATERIALS AND METHODS: Twenty-one male rats were randomly divided into 3 groups (n = 7): the control group received distilled water and the treatment groups received two doses of aqueous extract of Boswellia serrata gum resin (boswellia) (50 and 100 mg/kg) every day for 4 weeks. The animals were tested by the Morris water maze (MWM) and their hippocampus was isolated. Expression of CREB and BDNF genes was analyzed by Q-RT-PCR. RESULTS: The MWM test showed improvement in spatial learning and memory in both treatment groups. Gene expression analysis revealed a significant increase in BDNF but not CREB expression in rats treated with both 50 and 100 mg/kg doses in comparison with the control group. CONCLUSION: Although boswellia exerts its effects on memory formation at least partly by affecting the expression of BDNF, the results imply that boswellia probably affects memory via another BDNF-related pathway than the BDNF-CREB-BDNF cycle.

Single-strand conformational polymorphism analysis of a common single nucleotide variation in WRAP53 gene, rs2287499, and evaluating its association in relation to breast cancer risk and prognosis among Iranian-Azeri population.

The WRAP53 (WD40-encoding RNA antisense to p53) gene encodes an antisense RNA, essential for p53 stabilization and induction upon DNA damage. Single nucleotide polymorphisms (SNPs) in WRAP53 have been associated with risk of cancer, which strengthens the role of WRAP53 in the pathogenesis of human malignancies. In fact, WRAP53 has been considered as a candidate cancer susceptibility gene. Accordingly, we performed a study to examine the association of a frequent genetic variation in WRAP53, rs2287499 (C/G), with breast cancer risk and prognosis among Iranian-Azeri population. A case-control association study, including 206 cases and 203 controls from Iranian-Azeri population, was conducted. Genomic DNA was extracted from peripheral blood and tumor samples by salting-out method. SNP genotyping was carried out by polymerase chain reaction-based single-strand conformational polymorphism (PCR-SSCP) technique. The sequence variation of SSCP banding patterns was determined by sequencing. The collected data were analyzed through statistical package for the social sciences software, using Chi-square (χ (2)) or Fisher's exact tests, with a significance level of 0.05. No significant differences in the allele and genotype frequencies between cases and controls were detected. Similarly, no significant associations between genotypes and clinicopathological data were observed. Concisely, no significant overall associations between rs2287499 and breast cancer risk and prognosis were detected in the studied population. The rs2287499 SNP is not associated with breast cancer predisposition in Iranian-Azeri women; it also cannot be used as a molecular biomarker to predict breast cancer prognosis in Iranian-Azeri population.