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Objective: Nasopharyngeal Carcinoma (NPC) is an endemic head and neck malignancy in Asia Pacific regions that is associated with chronic infection by Epstein-Barr virus (EBV). EBV miR-BART-7 is a microRNA (miRNA) encoded by EBV that regulates malignant behavior of NPC. However, the role and function of miR-BART7 are not clear, particularly the relation of circulating levels and patient’s clinical presentation. Methods: Circulating miR-BART-7 levels were measured by using qRT-PCR and were correlated with clinical and pathological data. Result: Of 52 NPC patients included in this study, 85% were diagnosed in the late stages (Stage III-IV). 73% of tumors were non-keratinizing undifferentiated NPC, 92% of tumors were WHO class III histology and all cases were EBV-IgA positive. Over-expression of miR-BART7-3p was correlated with positive regional lymph nodes in newly diagnosed (4.61 fold changes, p

The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation. Here, we present a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures.

Background: Breast cancer is the leading cause of cancer mortality and morbidity among Indonesian women. Identification of biological pathways leading into therapeutic resistance through in vitro model is an important step to develop alternative effective therapy in breast cancer. Loss of PTEN expression has been associated with resistance to chemotherapy by involving PI3K/PTEN- dependent apoptosis pathway. We conducted in vitro experiment to investigate the association of hsa-miR-21 and PTEN expression in Doxorubicin-resistant MCF-7 cell line. Methods: Parental MCF-7 cells were periodically incubated with Doxorubicin to obtain specific Dox-resistant variant determined by IC50 using MTT assay. PTEN protein expression was analyzed using immunocytochemistry. Expression of mature has-miR-21 was measured using qRT-PCR. Results: The IC50 of Doxorubicin in parental MCF-7 and Doxorubicin-resistant MCF-7 cells (MCF-7/Dox) was 0.68 and 5.78 µg/ml, respectively. Hsa-miR-21 was significantly overexpressed in MCF-7/Dox cells compared to parental MCF cells (7.94 fold changes). Conclusion: PTEN and hsa-miR-21 expression levels were negatively correlated in Doxorubicin resistant-MCF cells. Further study to confirm the causal relationship of miR-21 overexpression and PTEN downregulation in MCF-7/Dox is required.

Doxorubicin (Dox)has beenused widely in breast cancer therapy. One of the problems in chemotherapy is the development of resistance to chemotherapy that lead to metastasis and relapse aggressiveness of cancer. MicroRNAs (miRNAs) are small non-coding RNA that regulate protein expression and play role in carcinogenesis, as well as cancer chemotherapy resistance. MiR-451 is classified as tumour suppressor miRNA, that binds to messenger RNA (mRNA) of MDR1, and leads disruption of P-glycoprotein (Pgp) expression. Thestudy aimed to investigate the association between miR-451 and Pgp related with Dox resistance mechanism. In silico analysis was conducted to predict the binding affinity between miR-451 and mRNA of MDR1. The MCF-7 cell line was used as wild type model, while MCF-7/Dox was used as a model of resistance. qPCR was conducted to calculated miR-451 expression and immunocytochemistry was used to observe Pgp expression. miRNA was down-regulated in both on MCF-7 and MCF-7/Dox. On the other hand, Pgp expression was detectable in the cytoplasmic and cytoplasmic membrane in MCF-7/Dox. The Pgp expression was higher in the MCF-7/Dox compared to MCF-7. In conlusion, the over expression of Pgp is associated with the resistance to MCF-7/Dox.

Breast cancer remains the leading cause of death among women, and there is a need to develop minimally invasive marker. In our previous study based on clinicopathologic in pre-chemotherapy patients showed miR-21 was upregulated 1.32 times higher at advanced stage compared with early stage. Therefore the matched patients for post-chemotherapy samples were used. The aim of this research is to examine the expression of miR-21 as potential marker to adjuvant chemotherapy in breast cancer patients. The samples were taken by using cross sectional method with total 39 blood plasma samples from breast cancer patients in adjuvant chemotherapy and 12 healthy control samples. Plasma was obtained from blood samples and then RNA isolated were performed. Total RNA was reverse transcribed using cDNA synthesis. The expression of miR-21 was then analyzed using specific primer for miR-21 and miR-16 as the reference gene. Livak Method was used to calculate the expression level in each group. The result showed that there is significant downregulated expression of miR-21 in postchemotherapy 2.61 fold compared with pre-chemotherapy (p

Epithelial Ovarian Cancer (EOC) is malignant cancer that caused death for most women in Indonesia. The emergence of EOC showed no specific symptoms in its early stages; that makes the screening mostly occur when patients are in advanced stage. Treatment of EOC at an advanced stage will be more challenging with poor prognosis. Therefore, minimally invasive biomarkers are needed to diagnose at the early stage. microRNA is one of the potential biomarkers which not only expressed inside the cell but also secreted outside the cell with exosome protection. This protection makes microRNA stable. Moreover, several studies have shown the ability to detect microRNA in the blood sample. microRNA-21 (miR-21) is oncomiR which targeted tumor suppressor mRNA RECK based on in silico analysis. The first aim is to determine the expression of miR-21 in plasma samples of EOC patients compared with healthy controls. The second aim is to investigate the expression correlation between miR-21 and RECK mRNA. Blood samples were collected from 30 patients and 30 healthy controls. Plasma was then obtained from centrifugated blood samples. The total RNA was isolated and reverse transcribed to produce cDNAs. cDNAs were then quantified using qPCR using specific primer for miR-21 and RECK mRNA. The expression analysis was done relative expression method by Livak. The expression of miR-21 was calculated using the miR-16 expression as the reference gene. Also, Beta-actin was used as reference gene for RECK mRNA calculation. The correlation between the expression of miR-21 and mRNA RECK was analyzed using the Spearman rho correlation analysis. In this study showed the expression of miR-21 in patients with EOC increased 4.7579-fold compared with healthy controls (p

Nasopharyngeal carcinoma (NPC) is the type of cancer related to multiple risk factors, including infection by Epstein Barr Virus (EBV). Standard treatment of NPC involves radiotherapy and chemotherapy in local and advanced tumors, while metastatic cases are treated with systemic chemotherapy. However, there is limited data on the causes of tumor recurrence, resistance, and progression. Moreover, the initial symptoms of NPC were often neglected until later enlarged, thus making it difficult to manage. MicroRNA (miRNA) is short molecule with 18-24 nucleotides and functions as protein-expression regulator protein in post-transcription. This study was aimed to determine miRNA expression and its relationship with the incidence of NPC. miR-21 and miR-29c were known to be involved in the development of NPC and resistance. A total of 51 plasma samples and 17 tissue samples were collected from Dharmais Hospital. The samples were taken from 17 untreated patients, 17 treated patients, and 17 healthy participants as control. We examined miRNA, protein of protein EBV (EBNA), and c-Myc expression using immunohistochemistry and quantitative polymerase chain reaction (qPCR). Our study revealed an increased expression of miR-21 and decreased expression of miR-29c in patients with NPC. There was also a correlation between the regulation of expression of miR-21 and c-Myc in the treated group of patients, and decreased expression in patients with complete response (CR) (4.13 ± 3.65: 2.74 ± 3.23; p

In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.

Breast cancer has emerged as the most common cancer-related mortality among women worldwide. Therefore, early cancer detection using biomarkers such as microRNA is needed. One of microRNAs that has an important role in breast cancer development is miR-155. Hsa-miR-155-5p is an oncomir that is commonly dysregulated in breast cancer. This study aims to determine the expression of hsa-miR-155-5p in breast cancer patient’s plasma before and after chemotherapy. We collected 64 samples from breast cancer patients admitted to Dr. Sardjito Hospital in Yogyakarta. RNA from plasma was extracted using RNA Isolation Kit miRCURY-Biofluid. cDNA synthesis was performed using cDNA Synthesis kit II and quantification of miR-155-5p using ExiLent SYBR Green master mix (Exiqon). qRT-PCR results were then analyzed with Livak's method and compared (before and after chemotherapy) with t-test. Expression of miR-155-5p in the breast cancer patients’ plasma after chemotherapy was significantly increased (10.59 times) when compared to before chemotherapy (p = 0.001). We concluded that there was upregulated expression of miR-155-5p after chemotherapy than before chemotherapy. There has not been a known, relevant pathway between hsa-miR-155-5p and chemotherapy regimens nor resistance to chemotherapy. Keywords: Breast cancer, plasma, hsa-miR-155-5p, oncomiR, chemotherapy.

SNPs of NQO1, p53, and MDM2 have been correlated with pathological response or overall survival of breast cancer patients treated with anthracycline based chemotherapy. Therefore, the ability to detect these SNPs may be useful for treatment decision. In this study, we genotyped FFPE breast cancer samples to detect those SNP. Objective The aim of this study was to know the accuracy of HRM (High Resolution Melting) method to genotype SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G's in comparison to RFLP (Restriction Fragment Length Polymorphism) from archived formalin fixed paraffin embedded (FFPE) breast cancer blocks. Materials and Methods FFPE blocks were collected from anatomic pathology laboratory. DNA were isolated, and PCR amplified followed by either RFLP or HRM methods. Restriction enzymes HinfI, BstUI, and MspA1I were used in RFLP to genotype NQO1, p53, and MDM2 SNPs, respectively. The primer design which been used was the same between HRM and RFLP. The results of HRM were analyzed using melting, normalization, and differentiation plot curves. Sequencing technique has been used to resolve discrepancy between RFLP and HRM results. Results We screened SNPs on breast cancer FFPE samples using HRM and RFLP. The rates of genotyping agreement between both methods are 69, 23% (9/13 samples), 80% (8/10 samples), and 100% (7/7 samples) for NQO1, p53, and MDM2 SNPs, respectively. Conclusion HRM is a faster method than RFLP to genotype important breast cancer SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G. Nevertheless, RFLP is more reliable method than HRM when the used primers sets are not optimized for HRM.