A new insight on serum microRNA expression as novel biomarkers in breast cancer patients.

Breast cancer (BC) is one of the widespread lethal diseases affecting a large number of women worldwide. As such, employing and identifying significant markers for detecting BC in different stages can assist in better diagnosis and management of the disease. Several diverse markers have been introduced for diagnosis, but their limitations, including low specificity and sensitivity, reduce their application. microRNAs (miRNAs), as short noncoding RNAs, have been shown to significantly influence gene expression in different disease pathologies, especially BC. Clearly, among different samples used for detecting miRNA expressions, circulating miRNAs present as promising and useful biomarkers. Among different body fluid samples, serum serves as one of the most reliable samples, thanks to its high stability under various severe conditions and some unique features. Extensive research has suggested that BC-related miRNAs can remain stable in the serum. The objective of this review is to describe different samples used for detecting miRNAs in BC subjects with emphasis on serum miRNAs. So, this study highlights serum miRNAs with the potential of acting as biomarkers for different stages of BC. We reviewed the possible correlation between potential miRNAs and the risk of early breast cancer, metastatic breast cancer, response to chemotherapy, and relapse.

Downregulation of HDAC2 and HDAC3 via oleuropein as a potent prevention and therapeutic agent in MCF-7 breast cancer cells.

Breast cancer is the most common malignancy in the world with the highest rate of morbidity and mortality. Due to the several side effects of chemotherapy and radiotherapy, recent studies have focused on the use of herbal medicines. Epidemiological reports have shown the inverse relationship between breast cancer risk and intake of olive. Oleuropein (OLE) is a polyphenolic compound in virgin olive oil with antineoplastic properties and it is well tolerated by humans. Recent reports have shown that OLE has effects on the control of cancer by modulating epigenetics, such as histone deacetylase (HDAC) inhibition. However, the epigenetic mechanisms of OLE anticancer properties are yet to be properly investigated. Therefore, this study aimed to determine the therapeutic effects of OLE through the modulation of histone deacetylase 2 (HDAC2) and histone deacetylase 3 (HDAC3) expression in breast cancer cell line. MCF-7 cells were tested with and without OLE, and also the cell viability, apoptosis, and migration were examined. HDAC2 and HDAC3 expression genes were assessed by quantitative real-time polymerase chain reaction. It was found that OLE decreased the expression of both HDAC2 and HDAC3 (P < 0.05), induced apoptosis and retarded cell migration and cell invasion in a dose-dependent manner (P < 0.05). These results showed that OLE is a potential therapeutic and preventive agent for breast cancer.

Narrower insight to SIRT1 role in cancer: A potential therapeutic target to control epithelial-mesenchymal transition in cancer cells.

The epithelial-mesenchymal transition (EMT) is a highly networked cellular process which involves cell transition from the immotile epithelial to the motile mesenchymal phenotype, whereby cells lose their cell-cell adhesion and cell polarity. This important process is one of the underlying mechanisms for enabling invasion and metastasis of cancer cells which is considered as malignant phase of tumor progression. However, the molecular mechanisms of this process are not fully clarified. It is reported that Sirtuin1 (SIRT1), a NAD+ dependent class III histone deacetylase is associated with tumor metastasis through positive regulation of EMT in several types of cancers. Recent studies confirmed that up and down regulation of SIRT1 expression remarkably change the migration ability of different cancer cells in vitro and tumor metastasis in vivo. Also, according to this fact that carcinomas as the main human solid tumors, originate from different epithelial cell types, SIRT1 role in EMT has received a great attention due to its potential role in tumor development and metastasis. Therefore, SIRT1 has been proposed as a key regulator of cancer metastasis by promoting EMT, although little is known about the cleared effect of SIRT1 in this transition. Our aim in this review is to explain in more detail the role of SIRT1 in various signaling pathways related to carcinogenesis, with the focus on the promoting role of SIRT1 in EMT as a potential therapeutic target to control EMT and to prevent cancer progression.

miR-449a: A Promising Biomarker and Therapeutic Target in Cancer and Other Diseases.

In the regulation of gene expression, epigenetic factors like non-coding RNAs (ncRNAs) play an equal role in genetics. The role of microRNAs (miRNAs), which are members of the ncRNA family, in post-transcriptional gene regulation is well-documented and has important implications for both normal and abnormal biological processes, such as angiogenesis, proliferation, survival, and apoptosis. The purpose of this study was to synthesize previous research on miR-449a by analyzing published results from various databases, as there have been a number of investigations on miR-449's potential involvement in the development of human disorders. Based on our findings, miR-449 is strongly dysregulated in a wide range of diseases, from various cancers to cardiovascular diseases, cognitive impairments, and respiratory diseases, and it may play a pivotal role in the development of these problems. In addition, miR-449a functions as a crucial regulator of the expression of several well-known genes, including E2F-3, BCL2, NOTCH1, and SOX4. This, in turn, modulates various pathways and processes related to cancer, including Notch, PI3K, and TGF-ß, and contributes to the improvement of cancer drug sensitivity. Curiously, abnormalities in the expression of this miRNA may serve as diagnostic or prognostic indicators for distinguishing between healthy people and patients or to evaluate the survival rates for specific disorders. This article provides a synopsis of the current understanding of miR-449a's role in human disease development through its regulation of gene expression and the biological processes related to these genes and their linked processes. In addition, we have covered the topic of miR-449a's potential as a clinical feature (diagnosis and prognosis) indicator for a range of disorders, both neoplastic and non-neoplastic. In general, our goal was to gain a thorough comprehension of the numerous functions of miR-449a in different disorders.

The impact of microRNAs on the resistance of breast cancer subtypes to chemotherapy.

Breast cancer (BC) formation is primarily influenced by genetics, epigenetics and environmental factors. Aberrant Genetics and epigenetics leads to a condition known as heterogeneity. The heterogeneity of BC can be divided into several subtypes. Among the epigenetic factors, microRNAs (miRNAs) have been shown to play a crucial role in the development and progression of malignancies. These small non-coding RNAs regulate gene expression through a variety of mechanisms, resulting in either mRNA degradation or translation repression. As miRNAs directly control many proteins, genetic anomalies affect tumor metastasis, apoptosis, proliferation, and cell transportation. Consequently, miRNA dysregulations contribute not only in cancer development but also in invasiveness, proliferation rate and more importantly, drug response. Findings mostly indicate subtype-specified identical miRNA profile in BC. Among the BC subtypes, TNBC, HER2 + and luminal are the most resistant to therapy, respectively. Therapy resistance is greatly associated with miRNA expression profile. Hence, concentration of miRNA is the first marker of its role in chemotherapy response. Overexpressed miRNAs may disrupt drug efflux transporters and decrease the drug accumulation in cell. While down-regulated miRNAs which mediate drug resistance processes are mostly correlated with poor treatment response. Moreover, other mechanisms in which miRNAs play crucial roles in chemoresistance such as cell receptor mediations, dysregulation by environmental factors, DNA defects, etc. Recently, several miRNA-based treatments have shown promising results in cancer treatment. Inhibition of up-regulated miRNAs is one of these therapeutic approaches whilst transfecting cell with down-regulated miRNAs also show promising results. Moreover, drug-resistance could also be determined while in the pre-treatment phase via expression levels of miRNAs. Therefore, miRNAs provide intriguing insights and challenges in overcoming chemoresistance. In this article, we have discussed how miRNAs regulate breast cancer subtypes-specific chemoresistance.

A novel missense variant in ESRRB gene causing autosomal recessive non-syndromic hearing loss: in silico analysis of a case.

BACKGROUND: Hereditary hearing loss (HHL) is a common heterogeneous disorder affecting all ages, ethnicities, and genders. The most common form of HHL is autosomal recessive non-syndromic hearing loss (ARNSHL), in which there is no genotype-phenotype correlation in the majority of cases. This study aimed to identify the genetic causes of hearing loss (HL) in a family with Iranian Azeri Turkish ethnicity negative for gap junction beta-2 (GJB2), gap junction beta-6 (GJB6), and mitochondrially encoded 12S rRNA (MT-RNR1) deleterious mutations. METHODS: Targeted genome sequencing method was applied to detect genetic causes of HL in the family. Sanger sequencing was employed to verify the segregation of the variant. Finally, we used bioinformatics tools and American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines to determine whether the detected variant might affect the corresponding protein or not. RESULTS: A novel homozygous missense mutation, c.499G>A (p.G167R), was identified in exon 5 of the ESRRB (estrogen-related receptor beta) gene. Healthy and affected family members confirmed the co-segregation of the variant with ARNSHL. Eventually, the variant's pathogenicity was confirmed by the in silico analysis and the ACMG/AMP guidelines. CONCLUSION: The study suggests that the detected variant, c.499G>A, plays a crucial role in the development of ARNSHL, emphasizing the clinical significance of the ESRRB gene in ARNSHL patients. Additionally, it would be helpful for genetic counseling and clinical management of ARNSHL patients and providing preventive opportunities.

The worldwide frequency of MYO15A gene mutations in patients with non-syndromic hearing loss: A meta-analysis.

MYO15A is the third most crucial gene in hereditary sensorineural hearing loss after GJB2 and SLC26A4. In the present study, we reviewed the prevalence of MYO15A mutations in patients with autosomal recessive non-syndromic hearing loss (ARNSHL). In this meta-analysis, we conducted a search of PubMed, Web of Science, Excerpta Medica Database, and Scopus, and identified the articles up to September 2019 without any time limit. Two investigators independently selected the relevant papers and extracted the required information. A total of 44 case-control and case series studies were considered, and 4176 patients and 3706 healthy individuals, as the control group, were included. The pooled frequency of MYO15A mutations between patients suffering from ARNSHL was calculated as 6.2% (95% CI: 4.9-7.8, P-value<0.001). There was heterogeneity between our studies (P-value<0.001, I2=58.1%); therefore, the random-effects model was utilized for analysis. Given the results, in many countries, the MYO15A gene has a significant contribution to hearing loss. Moreover, in several regions, specific dominant mutations in this gene have been reported. Therefore, the ethnic background should be considered to investigate the mutations of the MYO15A gene.

Differential Expression Pattern of Epithelial Mesenchymal Transition Gens: AXL, GAS6, Claudin-1, and Cofilin-1, in Different Stages of Epithelial Ovarian Cancer.

BACKGROUND: Epithelial ovarian cancer (EOC), is the fatal form of gynecological cancer. Almost 70% of ovarian cancer patients are detected at an advanced stage (III-IV) with metastases. Epithelial-mesenchymal transition (EMT) is a critical process associated with metastasis. This study investigated the expression levels of AXL, GAS6, Claudin-1, and Cofilin-1, as genes involved in EMT in relation to clinicopathologic features in ovarian cancer patients. METHODS: In this descriptive study, 78 ovarian epithelial cancer patients were enrolled. Samples were provided by the Iran National Tumor Bank, founded by the Cancer Institute of Tehran University of Medical Sciences in 2017. The expression levels of AXL, GAS6, Claudin-1, and Cofilin-1 genes were investigated in a fresh, frozen tumor sample and normal adjacent tissue by real-time PCR (RT-PCR). RESULTS: Findings showed a significant relationship between the overexpression of AXL and TNM staging (P=0.03). The expression level of GAS6 decreased in more advanced stages (P=0.01). There is a negative relationship between Cofilin-1 expression level and TNM staging (P=0.002). Claudin-1 expression level was higher in low stages compared with that in high stages (P=0.01). There was no relationship between gene expression levels of target genes with size and grade of the tumor. CONCLUSION: Given the importance of these genes in EMT, alteration in their expression pattern can contribute to the progression of the disease and distant metastasis of cancer cells. Additionally, knowing the alteration pattern of these genes expression can help to better understanding and prediction of the prognosis of EOC.

HDACis (class I), cancer stem cell, and phytochemicals: Cancer therapy and prevention implications.

Epigenetics is independent of the sequence events that physically affect the condensing of chromatin and genes expression. The unique epigenetic memories of various cells trigger exclusive gene expression profiling. According to different studies, the aberrant epigenetic signatures and impaired gene expression profiles are master occurrences in cancer cells in which oncogene and tumor suppressor genes are affected. Owing to the facts that epigenetic modifications are performed earlier than expression and are reversible, the epigenetic reprogramming of cancer cells could be applied potentially for their prevention, control, and therapy. The disruption of the acetylation signature, as a master epigenetic change in cancers, is related to the expression and the activity of HDACs. In this context, class I HDACs play a significant role in the regulation of cell proliferation and cancer. More recently, cancer stem cell (CSC) has been introduced as a minority population of tumor that is responsible for invasiveness, drug resistance, and relapse of cancers. It is now believed that controlling CSC via epigenetic reprogramming such as targeting HDACs could be helpful in regulating the acetylation pattern of chromatin. Recently, a number of reports have introduced some phytochemicals as HDAC inhibitors. The use of phytochemicals with the HDAC inhibition property could be potentially efficient in overcoming the mentioned problems of CSCs. This review presents a perspective concerning HDAC-targeted phytochemicals to control CSC in tumors. Hopefully, this new route would have more advantages in therapeutic applications and prevention against cancer.

An update of common autosomal recessive non-syndromic hearing loss genes in Iranian population.

Autosomal-recessive genes are responsible for about 80% of the hereditary non-syndromic hearing loss (NSHL) cases. In Iran, due to consanguineous marriages, NSHL is the second most frequent disability after intellectual disability, occurring one in 16 individuals. Enormous heterogeneity in the genetic pathology of hearing loss causes a major challenge in identification of responsible genes. In Iran, GJB2 is responsible for the most cases of pre-lingual and non-syndromic hearing loss (with frequency of 16.7%) which followed by other genes with lower frequency. Although several studies have indicated that a large proportion of both syndromic and non-syndromic hearing loss in Iranian populations are caused by defects in just a few genes, new detection strategies such as NGS (Next-generation sequencing) have increased the spectrum of responsible mutations. However, by applying this technique in Iran patients screening, the role of lots of novel related genes have been reported. In this review, we aim to describe function of these genes and their contribution to non-syndromic genetic hearing loss in Iranian population and we classify the genes by their functions.

Sodium Butyrate and Valproic Acid as Splicing Restoring Agents in Erythroid Cells of ß-Thalassemic Patients.

BACKGROUND: ß-Thalassemia is a common autosomal recessive disorder in human caused by a defect in ß-globin chain synthesis. The most common mutations causing ß-Thalassemia have been found to be splicing mutations. Most of which activate aberrant cryptic splicing/sites without complete disruption of normal splicing. IVSI-110 mutation, a common splicing mutation, leads to a 90% reduction of normal ß-globin synthesis and lead to blood transfusion dependency in the homozygote forms. However, modulation of splicing can be achieved by activation or suppression of transacting factors such as SR (Serine, Arginine) amino acids and hnRNPs (Heterogeneous ribonucleoprotein particle) through drugs. OBJECTIVES: The aim of this study was to investigate the effects of NaBu, isoBu and VPA drugs on restoration of splicing of IVSI-110 ß-Thalassemia pre-mRNA in human. MATERIALS AND METHODS: Primary erythroid cells derived from IVSI-110 ß-Thalassemia patients were cultured ex vivo and differentiated in the presence of 0.5 and 1 mM of Na-Butyrate (NaBu), 0.5 mM Isobutyramide (isoBu) and 100 µM Valproic acid (VPA). RT- PCR analysis was used to evaluate the effect of the drugs in correction of normal splicing in bglobin mRNAs. RESULTS: Following treatment with NaBu, isoBu and VPA, the level of normal ß-globin mRNA in Primary erythroid cells derived from IVSI-110 ß-Thalassemia patients, increased 1.7, 1.5, 1.4 fold, respectively relative to normal ß-globin mRNAs. Higher splicing restoration was achieved by NaBu, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. CONCLUSIONS: The results highlighted the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

Association study of IL2RA and CTLA4 Gene Variants with Type I Diabetes Mellitus in children in the northwest of Iran.

Introduction: A variety of genetic predisposing factors and environmental factors are known to influence the pathogenesis of type-1 diabetes (T1D). This study intended to investigate the association of cytotoxic T-lymphocyte associated protein 4 (CTLA4) and interleukin 2 receptor subunit alpha (IL2RA) gene polymorphisms with type 1 diabetes in children of northwest of Iran. Methods: Genomic DNA was extracted by salting-out method. PCR amplification and direct sequencing methods were used for genotyping of CTLA4 (exon 1) and IL2RA (intron 1) genes in all patients and controls. SNPStats was used to calculate odds ratios (ORs), 95% confidence intervals (CIs), and p values. Results: In this study, the frequency of G allele and GG genotype of CTLA-4 (+49A/G) polymorphism in T1D patients were significantly different from those in the controls (26% vs. 11%, p = 0.006). Moreover, a significant difference was observed between patients and control group in the allele frequencies of the new SNP (chr2:203868145) that was identified in exon one of CTLA4 (14% vs. 3%, p = 0.006). The results showed that the GG homozygous genotype of +49 A>G was associated with increased glycemic level in T1D patients in the study population (95% CI = 10.47, p = 0.0067). However, no significant association was found between IL2RA (ss52580101C>A) polymorphism and T1D patients (2% vs. 4%, p = 0.41). Conclusion: The results further support the association of T1D with +49A>G SNP in the CTLA4 gene in the population of northwest of Iran. However, no significant relationship was observed between ss52580101C>A polymorphism of IL2RA gene and T1D in this study.

Evaluation of Association Between HLA Class II DR4-DQ8 Haplotype and Type I Diabetes Mellitus in Children of East Azerbaijan State of Iran.

PURPOSE: Association between HLA-DR4-DQ8 haplotype and type 1 Diabetes Mellitus (DM-1A) was investigated in children of East Azerbaijan state of Iran because such an association has not been previously studied in this population. METHODS: HLA-typing was performed by polymerase chain reaction sequence-specific priming. For haplotype analysis, the logistic regression model was performed. RESULTS: Of the three investigated alleles, the frequency of DRB1*0401 was significantly higher among patients compared with that in healthy subjects (76.74% vs. 23.26%). CONCLUSION: The findings of the current study are consistent with those of previous studies and show that DRB1*0401 is associated with DM-1A; the frequencies of the two other alleles were also higher among patients, although the differences were not statistically significant. Two haplotypes associated with these alleles were also surveyed, and DRB1*0401--DQA1*0301-, and DRB1*0401--DQA1*0301--DQB1*0302- were the most frequent haplotypes among the patient group.

The Association between Human Leukocyte Antigen Class II DR3-DQ2 Haplotype and Type 1 Diabetes in Children of the East Azerbaijan State of Iran.

BACKGROUND: Type 1 diabetes mellitus (T1D) is an autoimmune disease. Several associations between human leukocyte antigen (HLA) complex and T1D were found in various populations. Associations with various HLA types depend on the investigated populations. However, such associations have not yet been investigated in the East Azerbaijan state of Iran with Turkish ethnicity. OBJECTIVES: The aims of the current study was to describe T1D genetic susceptibility conferred by HLA class II alleles (DRB1*0301, DQA1*0501 and DQB1*0201) and to determine haplotype frequencies among T1D patients. PATIENTS AND METHODS: This study was a case-control study. The number of samples was determined using the Cochran formula. Eighty unrelated T1D patients, including 42 (52.5%) females and 38 (47.5%) males, were randomly recruited from the East Azerbaijan state of Iran. Typing of HLA was performed by polymerase chain reaction-sequence-specific priming (PCR-SSP) on DNA extracted from peripheral blood mononuclear cells of 80 unrelated patients and 80 unrelated healthy control donors, who were selected randomly. For haplotype analysis, the logistic regression model was performed that allows joint estimation of Single-nucleotide polymorphisms (SNPs) via haplotypes. RESULTS: The frequency of drb1*0301 (82.5% vs. 11.3%), dqa1*0501 (82.5% vs. 36.3%) and dqb1*0201 (81.3% vs. 35%) were significantly higher among patients compared with that of healthy subjects. CONCLUSIONS: Our investigation demonstrated that there is a highly significant association between the studied alleles and T1D. It can be construed that haplotype HLA-DR3-DQ2 has a very modest effect with respect to the risk of T1D.

Minor role of BRCA2 mutation (Exon2 and Exon11) in patients with early-onset breast cancer amongst Iranian Azeri-Turkish women.

OBJECTIVE(S): Breast cancer is the most common cancer in women. Every year, one million new cases are reported worldwide, representing 18% of the total number of cancer in women. Hereditary BRCA1 and BRCA2 mutations account for about 60% of inherited breast cancer and are the only known causes of hereditary breast cancer syndrome. The aim of this study was to determine the frequency of BRCA2 (exon2 and exon11) gene mutation in patients with early-onset breast cancer among Iranian Azeri-Turkish women. MATERIALS AND METHODS: We obtained clinical information, family history and peripheral blood from 110 women under the age of 45 with early-onset breast cancer for scanning germline mutations in the exon2 and 11 of BRCA2 genes which comprises over 50% of the gene. Single-strand conformation polymorphism assay was used in order for screening potential mutations on amplified regions followed by direct sequencing analysis to determine the genotypes. RESULTS: Overall, 11 sequence variants were identified in this study group, including four homozygotes and seven heterozygotes silent substitution of c.3807T to C, p.Val1269Val (rs543304). CONCLUSION: Mutations in BRCA2 were surprisingly infrequent in the early onset breast cancer patients among Iranian Azeri-Turkish women.

Cloning of Soluble Human Stem Cell Factor in pET-26b(+) Vector.

PURPOSE: Stem cell factor (SCF) plays an important role in the survival, proliferation and differentiation of hematopoietic stem cells and progenitor cells. Potential therapeutic applications of SCF include hematopoietic stem cell mobilization, exvivo stem/progenitor cell expansion, gene therapy, and immunotherapy. Considering the cost and problem in accessibility of this product in Iran, clears the importance of indigenizing production of rhSCF. In the present work, we describe the construction of the soluble rhSCF expression vector in pET-26b (+) with periplasmic localization potential. METHODS: Following PCR amplification of human SCF ORF, it is cloned in pET-26b (+) vector in NcoI and XhoI sites. The recombinant construct was transformed into BL21 (DE3) Ecoli strains. RESULTS: The construction of recombinant vector was verified by colony PCR and sequence analysis of pET26b-hSCF vector. Sequence analyses proved that human SCF ORF has been inserted into NcoI and XhoI site with correct orientation downstream of strong T7 promotor and showed no nucleotide errors. CONCLUSION: The SCF ORF was successfully cloned in pET-26b (+) expression vector and is ready for future production of SCF protein.

The HLA-G 14bp insertion/deletion polymorphism in women with recurrent spontaneous abortion.

HLA-G is a non-classical HLA class Ib molecule with limited protein variability generated by alternative splicing. HLA-G displays immunotolerant properties and hence plays important roles in the maintenance of a successful pregnancy and maternal tolerance of the semiallogenic fetus. Polymorphism of the HLA-G gene may potentially affect the biological properties of the protein, and a 14-bp insertion/deletion polymorphism in exon 8 of the 3' untranslated region (3' UTR) of the HLA-G gene is thought to influence HLA-G expression. To study the association of the 14-bp insertion/deletion (INDEL) polymorphism with the risk of recurrent spontaneous abortion (RSA), we used polymerase chain reaction (PCR) amplification, and genotyped 85 women in the case group (women who have had two or more unexplained RSA) and 85 women in the control group (women who have had at least one normal pregnancy). Our results showed that the frequencies of the-14 bp/-14 bp and +14 bp/+14 bp genotypes were reduced in women with RSA, while that of the +14 bp/-14 bp genotype was significantly increased in RSA compared with the control group of normal fertile women; no significant differences in the allele frequencies of the HLA-G 14-bp polymorphism were observed. These results suggest a possible significance of the HLA-G 14-bp INDEL polymorphism in the outcome of pregnancy. However, further studies on other polymorphic sites in the 3 UTR and 5' UTR regions, as well as monitoring the serum HLA-G concentration are necessary in order to determine the potential implications of this marker in our population.

Construction of pPIC9 Recombinant Vector Containing Human Stem Cell Factor.

PURPOSE: Various cytokine regulates hematopoesis; they promote number of stages in stem cells biology such as proliferation, differentiation and endurance. Biological effects of SCF, as a hematopoietic cytokine; is triggered by binding to its ligand c-kit. Potential therapeutic applications of SCF include hematopoietic stem cell mobilization, exvivo stem/progenitor cell expansion, gene therapy, and immunotherapy. In this study we tried to construct of pPIC9 recombinant vector containing human SCF. METHODS: hSCF cDNA was amplified by PCR and both hSCF cDNA and pPIC9 as yeast expression vector (shuttle vector) digested by EcoR I and Xho I restriction enzymes. Subsequent the digestion reaction, ligation reaction was carried out. In order to verifying of pPIC9 recombinant vector containing hSCF, PCR and sequence analysis was performed. RESULTS: The construction of recombinant expression vector of pPIC9 containing hSCF cDNA was confirmed by sequencing method successfully. CONCLUSION: rhSCF/pPIC9 vector can be transformed into the Picha pastoris yeast as a eukaryotic host in order to produce human SCF at industrial scale.

Construction of Yeast Recombinant Expression Vector Containing Human Epidermal Growth Factor (hEGF).

PURPOSE: The objective of this study was construction of recombinant hEGF-pPIC9 which may be used for expression of recombinant hEGF in following studies. METHODS: EGF cDNA was purchased from Genecopoeia Company and used for PCR amplification. Prior to ligation, the PCR product and pPIC9 vector was digested with EcoRI and XhoI and ligated in pPIC9 vector and subjected to colony PCR screening and sequencing analysis. RESULTS: PCR amplification of EGF cDNA using recombinant hEGF-pPIC9 vector as template was concluded in amplification of 197bp fragment. Construction of recombinant hEGF-pPIC9 of EGf gene was verified by PCR and sequencing. CONCLUSION: Construction of Recombinant hEGF-pPIC9 was the primary stage for production and expression of EFG in the future study.