In silico design of a multi-epitope vaccine against HPV16/18.

BACKGROUND: Cervical cancer is the fourth most common cancer affecting women and is caused by human Papillomavirus (HPV) infections that are sexually transmitted. There are currently commercially available prophylactic vaccines that have been shown to protect vaccinated individuals against HPV infections, however, these vaccines have no therapeutic effects for those who are previously infected with the virus. The current study's aim was to use immunoinformatics to develop a multi-epitope vaccine with therapeutic potential against cervical cancer. RESULTS: In this study, T-cell epitopes from E5 and E7 proteins of HPV16/18 were predicted. These epitopes were evaluated and chosen based on their antigenicity, allergenicity, toxicity, and induction of IFN-γ production (only in helper T lymphocytes). Then, the selected epitopes were sequentially linked by appropriate linkers. In addition, a C-terminal fragment of Mycobacterium tuberculosis heat shock protein 70 (HSP70) was used as an adjuvant for the vaccine construct. The physicochemical parameters of the vaccine construct were acceptable. Furthermore, the vaccine was soluble, highly antigenic, and non-allergenic. The vaccine's 3D model was predicted, and the structural improvement after refinement was confirmed using the Ramachandran plot and ProSA-web. The vaccine's B-cell epitopes were predicted. Molecular docking analysis showed that the vaccine's refined 3D model had a strong interaction with the Toll-like receptor 4. The structural stability of the vaccine construct was confirmed by molecular dynamics simulation. Codon adaptation was performed in order to achieve efficient vaccine expression in Escherichia coli strain K12 (E. coli). Subsequently, in silico cloning of the multi-epitope vaccine was conducted into pET-28a ( +) expression vector. CONCLUSIONS: According to the results of bioinformatics analyses, the multi-epitope vaccine is structurally stable, as well as a non-allergic and non-toxic antigen. However, in vitro and in vivo studies are needed to validate the vaccine's efficacy and safety. If satisfactory results are obtained from in vitro and in vivo studies, the vaccine designed in this study may be effective as a therapeutic vaccine against cervical cancer.

The relevance of long noncoding RNAs in colorectal cancer biology and clinical settings.

Colorectal cancer (CRC) is one of the most frequent causes of cancer-related death worldwide. The prognosis of the malignancy and patient survival is commonly poor. Therefore, the discovery of pertinent biomarkers is essential to provide an accurate diagnosis and effective therapy. Newly, a group of noncoding RNAs named long noncoding RNAs (lncRNAs) has been found to involve in CRC development and progression. In this review, we highlighted the biological function of lncRNAs and reviewed their potentials as clinical tools in the CRC. A literature search of PubMed, EMBASE, MEDLINE, Web of Science, Scopus, and Cochrane Library using the MeSH terms "CRC," "long noncoding RNA," "lncRNA," and relevant was completed. The review included all articles that reported on the significance and role of lncRNAs in CRC development and clinical settings. All identified articles were cross-referenced for further articles, and any unavailable online were retrieved from hardcopy archive libraries. CRC-related lncRNAs could regulate a number of cellular processes, and their dysregulations have been suggested as potential biomarkers.

Identifying genotype profile of chronic hepatitis C infection in Southwest Iran.

BACKGROUND: Hepatitis C virus (HCV) infection is one of the most important risk factors for liver failure which can lead to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Approximately 170-200 million (almost 3% of the world's population) people have been reported to have HCV infection worldwide. HCV has six genotypes and multiple subtypes. HCV genotyping and identification of subtypes are critical steps for HCV vaccine development. MATERIALS AND METHODS: In this community-based study, we aimed to investigate the HCV genotypes in infected patients referring to the laboratory of Hajar Hospital of Shahrekord city (the capital of Chaharmahal and Bakhtiari Province) in Iran from November 21, 2016, to October 21, 2017. During 2016-2017, the sera were obtained from 2377 individuals referring to the laboratory of Hajar Hospital of Shahrekord, Iran. The anti-HCV antibody was tested for all sera by enzyme-linked immunosorbent assay test. Following HCV RNA isolation and cDNA synthesis, HCV genotype detection was performed by quantitative reverse transcription-polymerase chain reaction. RESULTS: Genotypes 3, 1a, and 1b were found in 28.6% (95% confidence interval [CI]: 17.0%-40.0%), 9.5% (95% CI: 2.1%-17.0%), and 3.2% (95% CI: 0.0%-7.6%) of the patients, respectively. In 5 patients (7.9%, 95% CI: 1.1%-14.8%), however, we did not observe any genotypes. We could not find any significant difference between the plasma viral load of infected patients and different genotypes. There was no significant difference either between age groups and genotypes (P > 0.05). CONCLUSION: The findings of the present study determined that HCV genotype 3 was the predominant genotype followed by the genotypes 1a and 1b in Chaharmahal and Bakhtiari Province.

Synthetic biology in various cellular and molecular fields: applications, limitations, and perspective.

Synthetic biology breakthroughs have facilitated genetic circuit engineering to program cells through novel biological functions, dynamic gene expressions, as well as logic controls. SynBio can also participate in the rapid development of new treatments required for the human lifestyle. Moreover, these technologies are applied in the development of innovative therapeutic, diagnostic, as well as discovery-related methods within a wide range of cellular and molecular applications. In the present review study, SynBio applications in various cellular and molecular fields such as novel strategies for cancer therapy, biosensing, metabolic engineering, protein engineering, and tissue engineering were highlighted and summarized. The major safety and regulatory concerns about synthetic biology will be the environmental release, legal concerns, and risks of the engineered organisms. The final sections focused on limitations to SynBio.

Diagnostic value of the candidate microRNAs in thyroid fine-needle aspiration biopsy (FNAB) samples.

Background Thyroid cancer (TC) is known to be the most common endocrine malignancy with an incidence rate which has increased by 2.3-fold over the past 30 years. Approximately, 30% of the thyroid fine-needle aspiration biopsy (FNAB) outcomes are indecisive. Moreover, researchers recognized multiple differentially expressed microRNAs (miRNAs) as candidate diagnostic markers for thyroid nodules. The purpose of this study was to identify thyroid tumor-associated miRNAs in FNAB with the capacity to be developed as unique biomarkers. Materials and methods According to the study design, a quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) was applied to evaluate the expression levels of nine miRNAs (Let7, miR-34a, miR-146b, miR-221, miR-151, miR-155, miR-181b, miR-222 and miR-375) among 224 FNA samples as the training set. Results The findings of this study revealed that miR-181b and miR-146b are the best predictors to diagnose benign thyroid FNA samples from malignant samples. However, the remaining miRNAs were co-expressed and had no significant effect on the predictor model. On the other hand, sensitivity and specificity of miR-181b and miR-146b were reported at 83.0%-83.0% and 83.0%-66.0%, respectively. Conclusions According to the results of this study, miR-146b and miR-181b might be considered as adjunct markers contributing to thyroid FNAB in tumor types. In addition, miR-146b and miR-181b were recognized as biomarkers for discriminating benign thyroid nodules from malignant ones. It is suggested that further prospective clinical trials be conducted to evaluate the accuracy of such findings in a larger cohort and determine the clinical uses.

Possible involvement of PI3K/AKT/mTOR signaling pathway in the protective effect of selegiline (deprenyl) against memory impairment following ischemia reperfusion in rat.

Short-term cerebral ischemia led to memory dysfunction. There is a pressing need to introduce effective agents to reduce complications of the ischemia. Involvement of PI3K/AKT/mTOR signaling pathway has been determined in the neuroprotective effect of various agents. Selegiline (deprenyl) possessed neuroprotective properties. In this study global ischemia/reperfusion was established in rats. Selegiline (5 mg/kg for 7 consecutive days) administrated via intraperitoneal route. Possible involvement of PI3K/AKT/mTOR signaling pathway was evaluated using qRT-PCR, immunohistochemistry and histophatologic evaluations in the hippocampus. Spatial memory was evaluated by morris water maze (MWM). Results showed that ischemia impaired the memory and ischemic rats spent more time to find hidden platform in the MWM. Ischemia significantly decreased levels of PI3K, AKT and mTOR in the hippocampus. Histopathologic assessment revealed that the percent of dark neurons significantly increased in the CA1 area of the hippocampus of ischemic rats. Selegiline improved the memory as ischemic rats spent fewer time to find hidden platform in the MWM. Findings showed that selegiline increased the level and expression of PI3K, AKT and mTOR as well as decreased the proportion of dark neurons in the CA1 area of the pyramidal layer of the hippocampus. We concluded that selegiline, partially at least, through increases the expression of PI3K, AKT and mTOR as well as decreases the percent of dark neurons in the hippocampus could improve the memory impairment following the ischemia in rats.

Biological and Clinical Relevance of Long Non-Coding RNA PCAT-1 in Cancer, A Systematic Review and Meta-Analysis

Long non-coding RNA (lncRNA) prostate cancer associated transcript 1 (PCAT-1) has been identified as a potential biomarker for the diagnosis and prognosis of various cancers. We performed this systematic review and meta-analysis to evaluate the role of dysregulation as well as the biological and clinical significance of lnc-PCAT-1 for predicting the malignancy status in several cancers. Two independent reviewers conducted an extensive search in electronic databases of Medline, Embase, Scopus, Web of Science and PubMed until the December of 2017. Five articles investigating the clinical significance of lncRNA PCAT-1, including 996 patients, were analyzed. Our results revealed that the increased PCAT-1 expression was related to overall survival (OS) (HR = 1.9, 95%CI: 1.13-3.18, P=0.015). Also, pooled results of the diagnostic data analysis demonstrated that PCAT-1 has a sensitivity of 0.59 and specificity of 0.66 for cancer diagnosis. Moreover, pooled area under curve was 0.62 (95% CI: 0.58­0.69). This meta-analysis revealed that lncRNA PCAT-1 could be served as a potential diagnostic and prognostic biomarker in various solid tumors.

Long Non-Coding RNAs in Thyroid Cancer: Implications for Pathogenesis, Diagnosis, and Therapy.

Thyroid cancer is a rare malignancy and accounts for less than 1% of malignant neoplasms in humans; however, it is the most common cancer of the endocrine system and responsible for most deaths from endocrine cancer. Long non-coding (Lnc)RNAs are defined as non-coding transcripts that are more than 200 nucleotides in length. Their expression deregulation plays an important role in the progress of cancer. These molecules are involved in physiologic cellular processes, genomic imprinting, inactivation of chromosome X, maintenance of pluripotency, and the formation of different organs via changes in chromatin, transcription, and translation. LncRNAs can act as a tumor suppressor genes or oncogenes. Several studies have shown that these molecules can interact with microRNAs and prevent their binding to messenger RNAs. Research has shown that these molecules play an important role in tumorigenicity, angiogenesis, proliferation, migration, apoptosis, and differentiation. In thyroid cancer, several lncRNAs (MALAT1, H19, BANCR, HOTAIR) have been identified as contributing factors to cancer development, and can be used as novel biomarkers for early diagnosis or even treatment. In this article, we study the newest lncRNAs and their role in thyroid cancer.

In vivo identification of novel TGIF2LX target genes in colorectal adenocarcinoma using the cDNA-AFLP method.

BACKGROUND AND STUDY AIMS: Homeobox-containing genes are composed of a group of regulatory genes encoding transcription factors involved in the control of developmental processes. The homeodomain proteins could activate or repress the expression of downstream target genes. This study was conducted to in vivo identify the potential target gene(s) of TGIF2LX in colorectal adenocarcinoma. METHODS: A human colorectal adenocarcinoma cell line, SW48, was transfected with the recombinant pEGFPN1-TGIF2LX. The cells were injected subcutaneously into the flank of the three groups of 6-week-old female athymic C56BL/6 nude mice (n = 6 per group). The transcript profiles in the developed tumours were investigated using the cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. RESULTS: The real-time RT-PCR and DNA sequencing data for the identified genes indicated that the N-terminal domain-interacting receptor 1 (Nir1) gene was suppressed whereas Nir2 and fragile histidine triad (FHIT) genes were upregulated followed by the overexpression of TGIF2LX gene. CONCLUSION: Downregulation of Nir1 and upregulation of Nir2 and FHIT genes due to the overexpression of TGIF2LX suggests that the gene plays an important role as a suppressor in colorectal adenocarcinoma.

Serum miRNAs as Biomarkers for the Diagnosis and Prognosis of Thyroid Cancer: A Comprehensive Review of the Literature.

BACKGROUND/OBJECTIVES: Thyroid cancer is the most common endocrine malignancy and accounts for 1% of cancers. In recent years, there has been much interest in the feasibility of using miRNAs or miRNA panels as biomarkers for the diagnosis of thyroid cancer. miRNAs are noncoding RNAs with 21-23 nucleotides that are highly conserved during evolution. They have been proposed as regulators of gene expression, apoptosis, cancer, and cell growth and differentiation. METHODS: The Directory of Open Access Journals (DOAJ), Google Scholar, PubMed (NLM), LISTA (EBSCO), and Web of Science were searched. RESULTS: The serum level of miRNAs (miRNA-375, 34a, 145b, 221, 222, 155, Let-7, 181b) can be used as molecular markers for the diagnosis and prognosis of thyroid cancer in the serum samples of patients with thyroid glands. CONCLUSIONS: Given that most common methods for the screening of thyroid cancer cannot detect the disease in its early stages, identifying miRNAs that are released in the bloodstream during the gradual progression of the disease is considered a key method in the early diagnosis of thyroid cancers.

Homeodomain Protein Transforming Growth Factor Beta-Induced Factor 2 Like, X-Linked Function in Colon Adenocarcinoma Cells

Background: TGIF2LX (transforming growth factor beta-induced factor 2 like, X-linked) is a homeodomain (HD) protein that has been implicated in the negative regulation of cell signaling pathways. The aim of this study was to investigate the possible functions of TGIF2LX in colon adenocarcinoma cells. Methods: The human SW48 cell line was transfected with cDNA for the wild-type TGIF2LX gene and gene/protein over-expression was confirmed by microscopic analysis, real time RT-PCR and Western blotting techniques. In vitro cell proliferation was evaluated by MTT and BrdU assays. After developing a colon tumor model in nude mice, immunohistochemical (IHC) staining of tumor tissue was carried out for Ki-67 (proliferation) and CD34 (angiogenesis) markers. To predict potential protein partners of TGIF2LX, in-silico analysis was also conducted. Results: Obtained results showed over-expression of TGIF2LX as a potential transcription factor could inhibit either proliferation or angiogenesis (P<0.05) in colon tumors. In-silico results predicted interaction of TGIF2LX with other proteins considered important for cellular development. Conclusions: Our findings provided evidence of molecular mechanisms by which TGIF2LX could act as a tumor suppressor in colon adenocarcinoma cells. Thus, this gene may potentially be a promising option for colon cancer gene-based therapeutic strategies.

Staphylococcus aureus Enterotoxin B Down-Regulates the Expression of Transforming Growth Factor-Beta (TGF-ß) Signaling Transducers in Human Glioblastoma.

BACKGROUND: It has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies. OBJECTIVES: We examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-ß). MATERIALS AND METHODS: A human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-ß transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using one-way analyses of variance (ANOVA) test. RESULTS: We reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 µg/mL and 2 µg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner. CONCLUSIONS: We suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-ß growth signaling and the reduction of tumor cell proliferation.

Transforming Growth Factor Beta-Induced Factor 2-Linked X (TGIF2LX) Regulates Two Morphogenesis Genes, Nir1 and Nir2 in Human Colorectal.

A member of homeodomain protein namely TGIF2LX has been implicated as a tumor suppressor gene in human malignancy as well as in spermatogenesis. However, to our knowledge, dynamic functional evidence of the TGIF2LX has not yet been provided. The aim of the present study was to investigate the human TGIF2LX target gene(s) using a cDNA-AFLP as a differential display method. A pEGFP-TGIF2LX construct containing the wild-type TGIF2LX cDNA was stably transfected into SW48 cells. UV microscopic analysis and Real-time RT-PCR were used to confirm TGIF2LX expression. The mRNA expressions of TGIF2LX in transfected SW48 cells, the cells containing empty vector (pEGFP-N), and untransfected cells were compared. Also, a Real-time PCR technique was applied to validate cDNA-AFLP results. The results revealed a significant down-regulation and up-regulationby TGIF2LX of Nir1 and Nir2 genes, respectively. The genes are engaged in the cell morphogenesis process. Our findings may provide new insight into the complex molecular pathways underlying colorectal cancer development.

Evaluation of SD-208, a TGF-ß-RI Kinase Inhibitor, as an Anticancer Agent in Retinoblastoma.

Retinoblastoma is the most common intraocular tumor in children resulting from genetic alterations and transformation of mature retinal cells. The objective of this study was to investigate the effects of SD-208, TGF-ß-RI kinase inhibitor, on the expression of some miRNAs including a miR-17/92 cluster in retinoblastoma cells. Prior to initiate this work, the cell proliferation was studied by Methyl Thiazolyl Tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays. Then, the expression patterns of four miRNAs (18a, 20a, 22, and 34a) were investigated in the treated SD-208 (0.0, 1, 2 and 3 µM) and untreated Y-79 cells. A remarkable inhibition of the cell proliferation was found in Y-79 cells treated with SD-208 versus untreated cells. Also, the expression changes were observed in miRNAs 18a, 20a, 22 and 34a in response to SD-208 treatment (P<0.05). The findings of the present study suggest that the anti-cancer effect of SD-208 may be exerted due to the regulation of specific miRNAs, at least in this particular retinoblastoma cell line. To the best of the researchers' knowledge, this is the first report demonstrating that the SD-208 could alter the expression of tumor suppressive miRNAs as well as oncomiRs in vitro. In conclusion, the present data suggest that SD-208 could be an alternative agent in retinoblastoma treatment.

Modulation of transforming growth factor­ß signaling transducers in colon adenocarcinoma cells induced by staphylococcal enterotoxin B.

Colorectal cancer (CRC) is a notable cause of cancer­associated mortality worldwide, making it a pertinent topic for the study of cancer and its treatment. Staphylococcal enterotoxin B (SEB), an enterotoxin produced by Staphylococcus aureus, has been demonstrated to exert anticancer and antimetastatic effects due to its ability to modify cell immunity and cellular signaling pathways. In the current study, SEB was investigated, including whether it exerts its growth inhibitory effects on colon adenocarcinoma cells. This may occur through the manipulation of a key tumor growth factor, termed transforming growth factor­ß (TGF­ß), and its signaling pathway transducer, Smad2/3. The human colon adenocarcinoma HCT116 cell line was treated with different concentrations of SEB, and cell number was measured using MTT assay at different treatment times. Smad2/3 RNA expression level was analyzed in untreated or SEB­treated cells using quantitative polymerase chain reaction, which indicated significant differences between cell viability and Smad2/3 expression levels. SEB effectively downregulated Smad2/3 expression in the HCT116 cells at concentrations of 1 and 2 µg/ml (P=0.0021 and P=0.0017, respectively). SEB concentrations that were effective at inhibiting Smad2/3 expression were correlated with those able to inhibit the proliferation of the cancer cells. SEB inhibited Smad2/3 expression at the mRNA level in a concentration­ and time­dependent manner. The present study thus proposed SEB as an agent able to significantly reduce Smad2/3 expression in colon cancer cells, provoking moderate TGF­ß growth signaling and the reduction of tumor cell proliferation.

Down-regulation of miR-135b in colon adenocarcinoma induced by a TGF-ß receptor I kinase inhibitor (SD-208).

OBJECTIVES: Transforming growth factor-ß (TGF-ß) is involved in colorectal cancer (CRC). The SD-208 acts as an anti-cancer agent in different malignancies via TGF-ß signaling. This work aims to show the effect of manipulation of TGF-ß signaling on some miRNAs implicated in CRC. MATERIALS AND METHODS: We investigated the effects of SD-208 on SW-48, a colon adenocarcinoma cell line. The cell line was treated with 0.5, 1 and 2 µM concentrations of SD-208. Then, the xenograft model of colon cancer was established by subcutaneous inoculation of SW-48 cell line into the nude mice. The animals were treated with SD-208 for three weeks. A quantitative real-time PCR was carried out for expression level analysis of selected oncogenic (miR-21, 31, 20a and 135b) and suppressor-miRNAs (let7-g, miR-133b, 145 and 200c). Data were analyzed using the 2-∆∆CT method through student's t-test via the GraphPad Prism software. RESULTS: Our results revealed that SD-208 could significantly down-regulate the expression of one key onco-miRNA, miR-135b, in either SW-48 colon cells (P=0.006) or tumors orthotopically implanted in nude mice (P=0.018). Our in silico study also predicted that SD-208 could modulate the expression of potential downstream tumor suppressor targets of the miR135b. CONCLUSION: Our data provide novel evidence that anticancer effects of SD-208 (and likely other TGF-ß inhibitors) may be owing to their ability to regulate miRNAs expression.

Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma.

BACKGROUND: Transforming growth factor-ß (TGF-ß) pathway is involved in primary tumor progression and in promoting metastasis in a considerable proportion of human cancers such as colorectal cancer (CRC). Therefore, blockage of TGF-ß pathway signaling via an inhibitor could be a valuable tool in CRC treatment. METHODS: To evaluate the efficacy of systemic targeting of the TGF-ß pathway for therapeutic effects on CRC, we investigated the effects of a TGßRI (TGF-ß receptor 1) or TßRI kinase inhibitor, SD-208, on SW-48, colon adenocarcinoma cells. In this work, in vitro cell proliferation was studied by methyl thiazolyl tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays. Also, the histopathological and immunohistochemical evaluations were conducted by hematoxylin and eosin, and Ki-67 and CD34 markers were stained, respectively. RESULTS: Our results showed no significant reduction in cell proliferation and vessel formation (170 ± 70 and 165 ± 70, P > 0.05) in treated SW-48 cells with SD-208 compared to controls. CONCLUSION: Our data suggested that SD-208 could not significantly reduce tumor growth and angiogenesis in human colorectal cancer model at least using SW-48 cells.