The discovery of genes that exhibit strong effects on metastatic pancreatic cancer

Pancreatic cancer is one of the deadliest cancers. Alterations in gene expression play an essential role during disease development. Also, variations in gene expression are involved in metastasis. Due to the complexity of gene expression alterations, however, it is still challenging to pinpoint and thus possibly target the functional reason behind the aggressiveness of pancreatic cancer. A high-throughput screening was conducted using the CRISPR-Cas9 whole- genome screening strategy to discover genes that might be essential for metastasis, to define processes that may have the potential to affect the metastatic progression of pancreatic cancer. Whole-genome CRISPR-Cas9 screening was conducted in two cell lines from the same parental cells but with different metastatic potential: S2-007 (highly metastatic) and S2-028 (lowly metastatic). The screening was performed, and samples were analyzed by MAGeCK analysis to select candidate genes. TYMS, MEN1, MYBL2 were selected based on their gene essentiality. In vitro validation was then conducted to confirm the gene essentiality in both cell lines. Cell proliferation, cell viability, soft agar colony formation assay was performed to narrow down the candidate. In vitro validation, MYBL2 showed the most promising potential as an essential gene in the metastatic pancreatic cancer cell. Wound healing assay and invasion assay were done to further validate MYBL2. Next, KEGG pathway analysis and Ingenuity analysis showed that MYBL2 interactions mainly in cell cycle network. Further experiments were performed which focused more on the effect of MYBL2 knockout in the cell cycle. FOXM1 gene knockout was then added to the experiments since FOXM1 plays a role in the G2/M phase. The double transduction was done, followed by cell sorting. Sorted cells were then cultured, and cell viability assay and cell cycle assay were performed. All knockouts were validated using Western blot. MYBL2 knockout showed inhibition in cell proliferation, cell viability, and soft agar colony formation assay in the S2-007 cell line. In addition, MYBL2 loss also showed invasion inhibition in S2-007. The double knockout FOXM1-MYBL2 affects cell viability more than the FOXM1 or MYBL2 single knockout. The cell cycle assay results demonstrated that the FOXM1- MYBL2, FOXM1, and MYBL2 knockout showed cell cycle arrest at G2/M in S2-007. Altogether the results showed that MYBL2 showed potential as an essential gene in the high metastatic pancreatic cancer cell line. However, the mechanism and the reason why MYBL2 showed essentiality only in the S2-007 cell line is still unclear. Therefore, more experiments still need to be conducted to achieve the goal of further elucidating the role of MYBL2 in metastatic pancreatic cancer

Over- and down-expression mir-29c and mir-21 after chemotherapy and radio-therapy in nasopharyngeal carcinomas and the down-regulating proteins encoding eipstein barr virus and c-Myc.

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 <0.1). The parameters tend to increase in patients with partial response (PR) (3.00 ± 5, 86 compared to 8.77 ± 8.43; p <0.5), while no significant difference in expression of miR-29c in patients with CR and PR was detected. We concluded that miRNA might be detected in the plasma of NPC patients, and miR-21 might become a useful biomarker to determine therapeutic outcome in NPC patients.Keywords: nasopharyngeal cancer; miRNA; biomarke

The Expression of hsa-miR-155-5p in Plasma Samples Of Breast Cancer Before And After Chemotherapy

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

DETEKSI SINGLE NUCLEOTIDE POLYMORPHISM NQO1 P187S, p53 P72R, dan MDM2 T309G DENGAN TEKNIK PCR-RFLP DAN PCR-HRM PADA SAMPEL FORMALIN FIXED PARAFFIN-EMBEDDED (FFPE) PASIEN KANKER PAYUDARA

Detection test for Single Nucleotide Polymorphism (SNP) recently carried out by PCR-RFLP. This method needs post-PCR procedure to determine the result. Discovery of real time PCR lead to the new method for SNP detection, called PCR-HRM. This alternative method was being analyzed to find better detection method. Detection of SNP NQO1 P187S, p53 P72R, and MDM2 T309G was carried out because they are having a relation on the metabolism response on anthracycline chemotherapy. Formalin Fixed Paraffin-Embedded (FFPE) samples are routine samples that collected in cancer hospital. The riches of this samples can be used as useable diagnostic samples. Nevertheless, quality of DNA which is isolated from FFPE�s sample is poor because of formalin. were There two aims of this research. First was to know the benefit of FFPE sample�s in SNP detection. Second was to know the benefot of PCR-HRM and HRM�s profile in SNP NQO1 P187S, p53 P72R, and MDM2 T309G�s detection. FFPE�s samples were collected from operation. The samples were DNA�s isolated, and amplified using PCR (Polymerase Chain Reaction) for NQO1, p53, dan MDM2. Restriction Fragment Lenght Polymorphism (RFLP) was conducted using restriction enzymes HinfI, BstUI, dan MspA1I, respectively for NQO1, p53, dan MDM2. The second method was PCR-High Resolution Melting (PCR-HRM). This method was done using real time PCR Corbett Rotor Gene. The results was annalyzed using melting curve, normalization curve, and differentiation plot curve.The comparison between PCR-RFLP and PCR-HRM has been done to detect three SNP. Sequencing technique has been used as standart technique if there are any differences found between PCR-RFLP and PCR-HRM. There are 6 differences found in comparison between PCR-RFLP and PCR-HRM. The PCRRFLP �results showed 66,67% similarity with sequencing�s results. FFPE�s sample can be used for SNP NQO1 P187S, p53 P72R, and MDM2 T309G� detection. HRM�s profile can be analyzed using three curves. There are melting curve, normalization curve, and differentiation plot curve. The comparison between PCR-RFLP and PCR-HRM has done to detect three SNP. PCR-RFLP more reliable method than PCR-HRM if primer which is used not specially design for PCR-HRM. PCR-HRM is a faster method than PCR-RFLP for SNP NQO1 P187S, p53 P72R, and MDM2 T309G�s detection

Circulation microRNA expression profiles in patients with complete responses to chemoradiotherapy in nasopharyngeal carcinoma

Background: Nasopharyngeal carcinoma (NPC) is endemic cancer in Southeast Asia with a relatively poor prognosis. Chemoradiotherapy is a primary treatment that advantages certain patients, particularly in the early stages. New predictive and prognostic biomarkers are required to guide and select the best treatment. Aims: To evaluate the circulation expression profile of microRNAs (miRNAs) associated with responses to chemoradiotherapy in nasopharyngeal carcinoma. Methods: Peripheral blood from 17 patients was collected before and after chemotherapy and radiotherapy. Differential expression circulating miRNAs were analyzed using microRNA Cancer Panels and were compared among patients with complete responses. Differential expression analysis using GenEx 7 Multid, statistic represented by GraphPad Prism 9. Alterations mechanism signaling pathways and biological function using IPA (Ingenuity Pathways Analysis). Results: Using microRNAs Cancer Plate consisting of 116 miRNAs, we identified ten circulating miRNAs that were differentially expressed in NPC patients after chemoradiotherapy. Unsupervised clustering and confirmation using qRT-PCR showed that miR-483-5p, miR-584-5p, miR-122-5p, miR-7-5p, miR-150-5p were overexpressed and miRNA are miR-421, miR-133a-3p, miR-18a-5p, miR-106b-3p, miR-339-5p were significantly downregulated after chemoradiotherapy (p < 0.0001). In addition, ROC analysis through AUC (Area Under Curve) with 99% confidence interval (CI) p value < 0.0001. Gene enrichment analysis of microRNAs and the targeted proteins revealed that the main involved pathways for chemoradiotherapy in NPC were cell death and survival signaling pathways. Conclusion: qPCR profiling in circulating blood compared before and after chemoradiotherapy in nasopharyngeal carcinoma can identify pathways involved in treatment responses. miR-483-5p, miR-584-5p, miR-122-5p, miR-7-5p, miR-150-5p, miR-421, miR-133a-3p, miR-18a-5p, miR-106b-3p, miR-339-5p are differentially regulated after chemoradiotherapy in NPC

Wolbachia genetic similarity in different insect host species: Drosophila melanogaster and Yogyakarta’s (Indonesia) Aedes aegypti as a novel host

Aedes aegypti as a novel host. Biodiversitas 23: 2321-2328. Wolbachia naturally presents in a large number of insects and other arthropod species. The Wolbachia strain wMel from Drosophila melanogaster has been stably transinfected into Aedes aegypti where it stops the mosquito host from being infected with medically important arbovirus like dengue. Consequently, Ae. aegypti infected with wMel have been released in Indonesia as a public health intervention against dengue. This study genetically compared wMel from Yogya field-caught D. melanogaster and the wMel in stably transfected Ae. aegypti used for field releases in Yogyakarta, Indonesia. The genetic similarity between wMel Wolbachia was evaluated by sequencing of Wolbachia surface protein (wsp) gene and some polymorphic genomic regions of insertion sites (IS) and variable number tandem repeats (VNTR) loci. The sequence of the Wolbachia surface protein (wsp) gene was 100% identical between hosts. There is no insertion sequence among specimens. The insertion sequence IS-WD1310 was identical between wMel from both hosts and among other strains, as well as the IS-WD516/7. The VNTR-141 period was identical within wMel from both hosts and among other strains, the VNTR-105 as well. Wolbachia Yogya field-caught D. melanogaster and Wolbachia strain wMel present in Ae. aegypti used for bio-control of dengue were genetically identical. These findings provide beneficial understanding to answer the public attention on safety issues, especially on the genetic similarity between Wolbachia strain in the natural and transfected hosts of this novel technology for dengue control