Volume 18, 2018

10 Articles

Gene Therapy & Molecular Biology

APRIL Gene Expression in a Cohort of Egyptian Acute Myeloid Leukemia Patients: Clinical and Prognostic Significance

Amira Ahmed Hammam Shereen Mohamed Elhoseiny MD, Raafat Mohamed Abdelfattah MD² and Rania El-Sayed Sheir MD

Pages: 1 - 13 | June 2018

Keywords: AML, APRIL, RTQ-PCR

Background: APRIL (A Proliferation Inducing Ligand) is a member of the tumor necrosis factor (TNF) family. It is essential for the survival of normal and malignant B lymphocytes. Increased expression of APRIL is noted in most of hematological malignancies and auto immune diseases. Patients and methods: We investigated the expression level of APRIL mRNA in 50 de novo acute myeloid leukemia (AML) patients, together with 20 healthy controls using a Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RTQ-PCR) with a specific aim of determining its relation to clinical features and laboratory findings at diagnosis and its impact on the response to therapy. Results: APRIL mRNA expression level was significantly higher in AML patients than in controls (p<0.001). APRIL expression level was significantly higher in patients who didn't achieve CR compared to those who achieved CR (p<0.001). Patients who didn't achieve CR also had higher TLC, lower platelets and older age than CR patients. The difference was statistically significant (p<0.001, p=0.047, p=0.019) respectively. APRIL levels showed significant positive correlation with TLC (r=0.743.p<0.001), with age (r=0.296, p=0.037)and a negative correlation with platelets count (r= -0.443,p=0.001) and no correlation with gender, Hb level, BM blast, HSM or LNs enlargement . Conclusion: Our study has shown that APRIL is overexpressed in AML patients, its level might serve as an indicator for disease progression. APRIL might be an indicator for poor prognosis and treatment resistance in AML patient; therefore, APRIL antagonists may represent a novel therapeutic approach for the treatment of AML.

siRNA delivery technology for cancer therapy: promise and challenges

Raziyeh Amini F.Karimi, F.Azizi Jalilian, R.Ezati, Keng-Liang Ou, Hossein Hossienkhani

Pages: 14 - 25 | June 2018

Keywords: Small interfering RNAs (siRNA), Cancer therapy, Delivery systems, Nanoparticles

Small interfering RNAs (siRNA) technology has shown great promise as a new class of therapeutics intervention for treatment of cancer and other diseases. Despite the remarkable biological process for sequence specific gene regulation, the major limitations of siRNAs-based therapeutics are their rapid degradation by serum nuclease, poor cellular uptake, and rapid renal clearance following systemic delivery, off-target effects and induction of immune responses. Many researchers have tried to overcome these limitations with developing nuclease-resistant chemically modified siRNAs and variety of synthetic and natural biodegradable lipids and polymers for siRNA delivery to enhance efficacy and safety profiles. An ideal siRNAs based delivery systems for cancer therapy must be clinically suitable, safe and effective. In this review, we introduce the greatest challenges in achieving efficient RNAi delivery, and discuss design criteria and various delivery strategies for cancer therapy, including chemical modifications, lipid based nanovectors, polymer-mediated delivery systems, conjugate delivery systems, and others.

NF-κB is Over-expressed in Breast Cancer Cell Lines, MCF-7 and MDA-MB-231, Following miR-590 Transfection

Azar Sheikholeslami Mohammad Nabiuni, Ph.D.2,Ehsan Arefian, Ph.D.3, Fatemeh Jamshidi-Adegani, Ph.D. candidate

Pages: 26 - 33 | June 2018

Keywords: breast cancer, microRNAs, transfection, NF-κB

Breast cancer is a heterogenous disease, considered as the most common malignancy in women worldwide. Despite all efforts on identifying cancer, there is no definite therapy yet and much more attempts in discovering cancer biology seems necessary. Immunology of tumors declares the relevance of immune system, chronic inflammation and cancer. Many studies have supported the role of NF-κB in linking inflammation and tumorigenesis. NF-κB as an important actor of Inflammation, plays diverse roles in cancer. microRNAs have been implicated in a number of diseases including a broad range of cancers. miR-590 has been reported to regulate different pathways and affect different types of cancers. Objectives: Regarding that miR-590 plays various roles in different cells and it is down regulated in many cancer cells, we decided to transfect it in MDA-MB-231 (highly invasive) and MCF-7 (poor invasive) cells and trace the expression of NF-κB as an effective gene in cancer cells. The effect of miR-590 transfection in breast cancer cell lines on the expression of NF-κB was evaluated in this study. Materials and Methods: The primer for miR-590 was designed and used for PCR. The PCR product was extracted and the miRNA gene was cloned into the vector. miR-590-pLenti-III-eGFP vector was transfected into the breast cancer cells. 72 hours after transfection, the expression of miR-590 and NF-κB were measeured by qRT-PCR. Results: miR-590 was over-expressed as it was expected to occur and NF-κB level showed significant increase 72 hours after miRNA transfection in both cell lines. Conclusion: In this study, the sudden and redundant increase of NF-κB followed by miR-590 transfection in breast cancer cells was seen. It seems that miR-590 regulates NF-κB expression directly or indirectly. miR-590 sounds to be an effective regulatory factor in breast cancer which requires more studies to investigate its function on cancer and inflammation.

Forkhead box p3 (Foxp3) gene polymorphisms and risk of unexplained recurrent spontaneous abortion among Egyptian women

Hanan Mohamed Farhan Dr Maha Hassan Katta

Pages: 34 - 42 | June 2018

Keywords: URSA, Foxp3 gene polymorphism, Tregs, PCR-RFLP

Transcription factor forkhead box p3 (Foxp3) gene plays crucial role in T regulatory (Treg) cells development and function. Tregs are involved in mediating maternal tolerance to the foetus and avoiding immunological rejection of the foetus. Our study aimed to determine whether there is a relationship between genetic polymorphisms of Foxp3 and susceptibility to unexplained recurrent spontaneous abortion (URSA). Materials and Methods: Using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) assay, we examined the frequency of Foxp3, rs3761548AC and rs2294021TC, gene polymorphisms in Egyptian URSA patients (n = 40) and control individuals (n = 40). Results: Foxp3 rs3761548AC (p = 0.012) and rs2294021TC (p<0.001) genotype polymorphisms were significantly more frequent in women with URSA than in controls with odds ratio (OR) values 6.3 (95%CI 1.3-31.1) and 9.3 (95%CI 3.3–26.1), respectively. There were differences in the distribution of A allele of rs3761548AC (OR=1.5, 95%CI=0.8-2.9, p=0.197) and T allele of rs2294021TC (OR=4, 95%CI=1.9-8, p<0.001) between URSA and controls; where only T allele reached statistical significance. Conclusion: Foxp3, rs3761548AC and rs2294021TC, gene polymorphisms may be considered as genetic risk factors in development of URSA in Egyptian patients.

Tumor Necrosis Factor Gene Polymorphisms and their Association with Susceptibility to Rheumatoid Arthritis, Disease Activity and Severity

Rabab A. Mohamed Heba M. Zagloul, Sanaa A. Elshafy, Enas A. Abdelaleem, Rania E. Sheir, Heba H. El-Demellawy

Pages: 66 - 77 | June 2018

Keywords: Rheumatoid arthritis (RA), genetic susceptibility, TNF polymorphisms, Polymerase chain reaction, Restriction fragment length polymorphism (PCR-RFLP).

Rheumatoid arthritis (RA) is a chronic autoimmune disease that shows multifactorial inheritance resulting from a complex interplay between an individualÂ’s environmental and genetic background. Tumor necrosis factor (TNF), a pro-inflammatory cytokine that has been shown to play a central role in the pathogenesis of numerous autoimmune diseases including RA. Aim: The present case control study was conducted to assess the association of LTA252A>G, TNFα-308G>A and TNFα-1031T>C gene polymorphisms with RA and their involvement in disease activity and severity. Methods: PCR-RFLP was used to detect the association of LTA252A>G, TNFα-308G>A and TNFα-1031T>C gene polymorphisms with RA. Results: TNF-α 308 G allele and TNF –α 308 GG genotype were significantly higher in RA patients compared to healthy control subjects (P=0.04; P=0.001, respectively). TNF-α 308 G allele and GG genotype were significantly higher in the RA non-remission group (P=0.008; P<0.001, respectively) compared to the remission group .On analysis of disease severity ,the TNF-α 308 AG genotype was more frequently represented among RA patients compared to GG and AA genotypes (P=0.007). There was no significant association between LTA252A>G and TNFα-1031T>C gene polymorphisms and RA. Conclusion: Our results suggest that TNF-α 308 G/A gene polymorphism is genetically associated with Rheumatoid Arthritis and involved in disease activity and severity in the Egyptian population.

Gene therapy to treat obesity

Dwaipayan Sen, PhD Sayani Sahu, PallaviSinha Roy, Konkona Datta, Gundu H.R. Rao

Pages: 78 - 88 | June 2018

Keywords: Obesity, Gene Therapy, CLOCK, adeno associated vector, CRISPR/Cas9, weight gain, adipocytes

This article focuses on reviewing the significant genes that can be regulated in order to potentially cure obesity. The notable targets discussed in this article include: 1) polymorphic sites on Period 3 (PER3)/Circadian Locomotor Output Cycles Kaput (CLOCK )/Rev-Erb-alpha along with associated regulatory sites on AANAT/CSNK1E genes which influence both circadian cycle and obesity; 2) beta-adrenergic receptor genes that play an important role in thermogenesis, thereby promoting lipolysis; 3) Peroxisome Proliferator Activated Receptors (PPAR) gene encoding PPAR that regulates adipocyte differentiation; 4) Fat mass and obesity associated gene (FTO) involved in food intake and fatty acid metabolism; 5) Low Density Lipoprotein (LDL) receptors that maintain plasma lipid levels through cholesterol metabolism, and 6) glucocorticoids receptor that induces adipocyte differentiation thus depositing visceral fat. Any polymorphic differences existing in these genes of various ethnic groups can possibly explain the prominent difference in the fat distribution patterns, which are most evident among Asians and Caucasians that can potentially be mitigated by gene therapy techniques. However since obesity is not a ‘single gene’ defect, it requires a detailed mapping of their genetic pathway and their association with other metabolic pathways along with careful selection/validation of the gene therapy tools before successful clinical translation.

Possible role of microRNA-122 in modulating multidrug resistance of hepatocellular carcinoma

Shaymaa M.M. Yahya Shadia A. Fathy, Zakaria A. El-Khayat, Safinaz E. El-Toukhy3, Ahmed R. Hamed4, Marwa G.A. Hegazy and Heba K. Nabih

Pages: 89 - 102 | June 2018

Keywords: Hepatocellular carcinoma; miRNA-122;MDR; ABCs; cell cycle

Hepatocellular carcinoma (HCC) is a hypervascular primary liver cancer characterized by rapid progression , besides, resistance to traditional chemotherapeutic agents. It has been shown that microRNAs play critical roles in regulation of tumor cell sensitivity to drugs. The present study investigated whether restoration of mir-122 in HCC cells could alter the cell cycle distribution and the expression of multidrug resistance (MDR)-related genes (ABCB1, ABCC1, ABCG2 and ABCF2). After overexpression of mir-122 in HepG2 cells treated or untreated with doxorubicin doses, total RNAs and protein extracts were isolated for application of QRT- PCR and western blotting techniques. Moreover, cell cycle distribution was monitored by flowcytometry. Our results revealed that, the over expression of mir-122 in HepG2 cells treated or untreated with doxorubicin could modulate the sensitivity of cells to chemotherapeutic drug through downregulation of MDR-related genes, ABCB1and ABCF2. The anti-proliferative effect of mir-122 is associated with the accumulation of cells in G0/G1 phase. Moreover, treatment with mir-122 and doxorubicin resulted in high percentage of HCC cells in G0/G1 phase. Taken together, our findings revealed that, overexpression of mir-122 inhibited HCC cell growth by inducing cell cycle arrest and this arrest is associated with down-regulation of MDR-related genes.

The effect of newly synthesized heterosteroids on miRNA34a, 98, and 214 expression levels in MCF-7 breast cancer cells

Shaymaa M.M. Yahya Gamal A. Elmegeed, Mervat S. Mohamed, Rafat M. Mohareb, Mervat M. Abd-Elhalim, Ghada H. Elsayed

Pages: 103 - 111 | June 2018

Keywords: Breast cancer; Steroids; Cytotoxicity; Apoptotic genes; miR-34a, miR-98; miR-214.

Hybrid anticancer drugs have emerged as great therapeutic captions that can effectively overcome most obstacles facing conventional anticancer drugs. miRNAs are considered as class of non-coding RNAs that can negatively regulate protein coding gene expression. miRNA expression is commonly altered in cancer cells. The current work aimed to test the effect of new pro-apoptotic heterosteroids on some drug resistance related miRNAs expression levels (miRNA34a, 98, and 214 ) in MCF-7 breast cancer cells. After cell treatment with these compounds 4, 6, 7, 13, 18, 21, 22 and 24, miRNAs were extracted and subjected to reverse transcription and subsequent PCR amplification using Real Time-PCR technique. The expression levels of miR-34a, miR-98 and miR-214 were quantitatively determined. The study revealed that the expression levels of miR-34a, miR-98 and miR-214 were up-regulated upon treatment with tamoxifen, which was used as a positive control drug , as compared to control cells,. Strikingly, the levels of miR-34a, miR-98 and miR-214 expression significantly down-regulated when treated with most of the new heterosteroids as compared to control cells. These results could indicate the promising effects of these new heterosteroids on reducing drug resistance as compared to tamoxifen drug. As well established, cells develop drug resistance to tamoxifen.

CRISPR: A New Era In Molecular Biology

Sumiyah Rasool Tufail Hussain, Asima Zehra, Shafat Khan, Shafqat Khan

Pages: 112 - 118 | June 2018

Keywords: CRISPR, bacterial immunity, gene editing

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. Editing via (CRISPR)–CRISPR-associated (Cas) constitutes a next-generation method for programmable and highthroughput functional genomics. CRISPR–Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Bacteria and archaea acquire resistance to invading viruses and plasmids by integrating short fragments of foreign nucleic acids at one end of the CRISPR locus. CRISPR loci are transcribed and the long primary CRISPR transcript is processed into a library of small RNAs that guide the immune system to invading nucleic acids, which are subsequently degraded by dedicated nucleases.

Overexpression of RhoGDI2 in stage II and III gastric cancer is involved in 5-fluorouracil resistance

Wang Jinguo Hu Minhua, Wu Pei and Wu Jian

Pages: 119 - 130 | June 2018

Keywords: RhoGDI2, 5-fluorouracil, chemotherapy, gastric cancer

Rho GDP dissociation inhibitor 2 (RhoGDI2) is known to be involved in tumor growth, metastasis and cisplatin resistance. We have further explored RhoGDI2 as a predictor of 5-fluorouracil (5-FU) treatment response in gastric cancer. Here we report that RhoGDI2 promotes tumor progression and 5-FU chemoresistance in gastric cancer. In clinical analysis, RhoGDI2 was overexpressed in 153 of 215 (71.16%) stage II and III patients who received 5-FU-based chemotherapy after radical gastrectomy. Multivariate Cox regression analysis indicated that RhoGDI2 expression was an independent prognostic factor for overall survival (P=0.013) and disease-free survival (P=0.008) of patients with gastric cancer. After being treated with 5-FU, the expression of RhoGDI2 in gastric cancer cells was increased. In RhoGDI2-overexpressing cells treated with 5-FU, cell viability decreased slower than in control cells. These findings demonstrate that RhoGDI2 is an independent prognostic factor for adjuvant 5-FU-based chemotherapy in patients with stage II and stage III gastric cancer. Targeting RhoGDI2 may thus be a useful strategy to enhance chemotherapy efficacy in gastric cancer.