MicroRNA Gene Signature for Predicting Mechanisms in Nasopharyngeal Carcinoma: A Case Study on the Potential Application of Circulating Biomarkers.

BACKGROUND AND AIM: Nasopharyngeal Carcinoma (NPC) is an upper respiratory tract cancer prevalent in Southeast Asia and related to chronic EBV infection. microRNAs (miRNAs) regulate gene expression implicated in NPC's carcinogenesis. However, this circulating RNA molecule's role and clinical utility remain unknown. Therefore, this study examined the circulation of miRNAs and their association with clinical data. METHODS: 160 plasma samples of NPC and 80 non-tumor samples were extracted to evaluate and validate the gene expressions. Quantification expression was performed using relative quantification of qPCR analysis level expression methods. The intrinsic cellular roles involving biological signaling in NPC's oncogenesis using Ingenuity Pathways Analysis (IPA) were also used. RESULTS: The results of the quantification significance profiling of NPC samples revealed decreased miR- 29c-3p (fold change 1.16; p<0.05) and increased 195-5p expression (fold change 1.157; p<0.05). Furthermore, the validation of hsa-miR-29c-3p expression on plasma NPC with known tumor vs. non-tumor and significant changes was also performed using a fold change of 4.45 (medians of 31.45 ± 1.868 and 24.96 ± 1.872, respectively; p<0.0005). miR-29c had a 2.14 fold change correlated with T primary status with a median of 31.99±1.319 and 31.35±2.412, respectively (p<0.05). Stage status with fold change 1.99 also had median levels of 31.98±1.105 and 31.21 ± 2.355, respectively (p-value <0.05). Furthermore, the node's status for the lower expression of miR-29c with fold change 1.17 had median levels of 32.78 ± 2.221 and 31.33 ± 1.689, respectively (p-value of 0.7). Bioinformatics analysis established the roles and functions of miR-29 in NPC progression, cell death and survival, cellular development, cellular function, and cell maintenance by inhibiting COL4A, PI3K, VEGFA, JUN, and CDK6. CONCLUSION: Overall, we conclude that decreased miR-29c expression is associated with poor clinical status and might inhibit NPC's five target genes.

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.

Potential plasma biomarkers: miRNA-29c, miRNA-21, and miRNA-155 in clinical progression of Hepatocellular Carcinoma patients.

This study evaluated differences in the clinical appearance of patients with hepatocellular carcinoma (HCC) based on plasma level and regulation of microRNAs (miRNA-29c, miRNA-21, and miRNA-155). The observational-analytical study with a cross-sectional design was conducted on 36 HCC patients and 36 healthy controls. The blood samples were collected from 2 Province Hospitals (Dr. Sardjito Hospital and Prof. Dr. Margono Soekarjo Hospital) for HCC and the Blood Bank Donor of the Indonesian Red Cross for 36 healthy controls. These blood samples were treated as follows: plasma isolation, RNA isolation, cDNA synthesis, quantification by qRT-PCR using a sequence-specific forward primer, and normalization of miRNA using housekeeping-stably miRNA-16. There were only 27 HCC patients with complete clinical variables (neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), platelet count, albumin, C-reactive protein (CRP), and cholinesterase (ChE)) that were able to analyses for regulation miRNAs based on its fold change expression miRNA target. All 27 HCC subjects were follow-up until 3-years of monitoring for their overall survival. The miRNA plasma expression was analyzed by Bio-Rad CFX 96 Manager software to determine the cycle of quantification, followed by the calculation of expression levels using Livak's methods. Data were analyzed using STATA 11.0, with a significant value of p<0.05. The miRNAs expression of HCC subjects were lower than that healthy control subjects in miRNA-29c (down-regulation 1.83-fold), higher than that healthy control subjects in miRNA 21 and miRNA-155 (up-regulation, 1.74-fold; 1.55-fold) respectively. NLR, CRP, ChE, and platelet count showed a significant difference in miRNA-29c regulation, though neutrophil count showed a significant difference in miRNA-21 and miRNA-155 regulation (p<0.05). Conclusion: Plasma biomarkers: miRNA-21 and miRNA-155 might be potential biomarkers as onco-miR in HCC subjects, while miRNA-29c might act as a tumor suppressor. Significant evidence was identified with clinical progression based on the regulation of miRNAs, which was consistent with miRNA -29c.

Transcriptome Profile of Next-Generation Sequencing Data Relate to Proliferation Aberration of Nasopharyngeal Carcinoma Patients in Indonesia.

OBJECTIVE: Nasopharyngeal carcinoma (NPC) is the most common cancer arising from epithelial cells of the nasopharynx in  Indonesia. This study aims to determine the differential level of gene expression in NPC patients when compared with normal individuals. Transcriptome profiling analysis was performed using RNA-Seq technology to determine the differential gene expression relate to proliferation aberration that occurs in NPC patients compared with normal individuals. So we get the transcriptomic profile of Indonesia NPC patients. METHODS: In this study, we used 9 samples, 7 NPC samples and 2 normal samples as control.  Fresh tissue of tumor samples was collected from biopsy, and normal samples were collected brushing technique. The total RNA was isolated from fresh tissue samples and brushing samples using the Rneasy® RNA Extraction Mini Kit. The cDNA library was generated using TruSeq® RNA Library Preparation Kit V2, and its concentration was determined using qPCR. The library was sequenced using the Next-Generation Sequencing (NGS) Illumina Next Seq 550 platform. The raw sequence data quality was analyzed using FastQC and interpreted using HISAT2, HTSeq, edgeR, and PANTHER. RESULTS: From the analysis, 25493 gene transcripts were expressed, with 1956 genes were significantly upregulated, 90 genes were significantly downregulated in NPC samples, and 23897 genes didn't change the expression level significantly (p <0.05), 10 of which genes were associated with cell proliferation. These genes are involved in the regulation of cancer cell proliferation through several signaling pathways, which are the apoptosis signaling pathway, IGF signaling pathway, Notch signaling pathway, and P13K signaling pathway. CONCLUSION: There were significant differences in gene expression levels between NPC patients and normal individuals. Each gene that has changed the expression level plays a role in regulating various pathways that lead to cell proliferation aberration in NPC cases.

Transcriptome Related to Avoiding Immune Destruction in Nasopharyngeal Cancer in Indonesian Patients Using Next-Generation Sequencing.

OBJECTIVE: This study aims to obtain the transcriptomes profile associated with avoiding immune destruction from nasopharyngeal cancer patients in Indonesia using next-generation sequencing. METHODS: The samples are divided into two types of samples; 1) biopsy of nasopharyngeal cancer tissue samples, 2) brushing tissue of people without nasopharyngeal cancer as control samples. The sequencing results were mapped (HISAT2) and quantified (HTSeq) for differential expression analysis using edgeR software. Transcripts data analyzed with Pantherdb and DAVID software to find genes related to the immune system and pathways related to immune destruction by cancer. RESULTS: The differential expression results show that 2,046 genes that have a significant differential expression. The 90 genes expression has down-regulated and 1,956 genes expression up-regulated, there are 20 genes related to the immune system. The 20 genes related to the immune system by analyzing lionproject.net that directly related to hallmark avoiding immune destruction that genes are CXCL9/10/11. The gene expression of CXCL9/10/11 regulates PD-L1 expressions via the Jak/STAT signaling pathway. The interaction between the extracellular domain PD-1 and PD-L1 in cancer cells have avoiding immune destruction. CONCLUSION: The results of this study suggest that the gene expression of CXCL9/10/11 have up-regulated is related to avoiding immune destruction that can use as an early detection biomarker of nasopharyngeal cancer in Indonesian patients.

The Effects of Combination of Mimic miR-155-5p and Antagonist miR-324-5p Encapsulated Chitosan in Ovarian Cancer SKOV3.

OBJECTIVE: Ovarian cancer is a malignant tumor that attacks reproductive organs of women. MicroRNA is known to have an involvement in the prognosis of ovarian cancer. One of them is miR-155-5p which is down regulated and miR-324-5p which is up regulated. Chitosan is used as microRNA delivery system. The aims of this study is to find out the effects of combination microRNA encapsulated chitosan in cell line SKOV3. METHODS: Cell line SKOV3 obtained from Stem Cell and Cancer Institute (Kalbe). Mimic miR-155-5p and Antagonist miR-324-5p formulated with chitosan. Total RNA was extracted from nine samples (three as control and six as treatment), and prepared for cDNA synthesis. Expression of RNA and mRNA target was measured using q-PCR Biorad CFX96 C.100 and Gen Ex 7 software. Statistics analysis was measured using SPSS 16.0. RESULTS: The administration of combination microRNA encapsulated with chitosan affect the expression of miR-155-5p and miR-324-5p endogen (p <0.05). The expression of mRNA target HIF1α and GLI1 was down regulated after treatment. The correlation between expression of microRNA and mRNA target was strongly (p <0.05). CONCLUSION: This study successfully presented effects of combination of mimic miR-155-5p and antagonist miR-324-5p encapsulated chitosan which be considered as a potential therapy targets for ovarium cancer.

Transcriptome Profile of Next Generation Sequence Data Related to Inflammation on Nasopharyngeal Carcinoma Cases in Indonesia.

OBJECTIVE: Transcriptomic Profile Analysis Related to Inflammation in Nasopharyngeal Carcinoma Cases. METHODS: This study used 2 control samples taken using the brushing technique and 7 cancer samples with tissue biopsy. Isolate total RNA using Rneasy® RNA Extraction Mini Kit. Measurement of total RNA concentration and purity using a fluorometer and nanodrop Qubit. Synthesis of cDNA library uses TruSeq® RNA Library Preparation Kit V2 and concentration is measured using qPCR. Sequencing samples using NGS Illumina NextSeq 550 platform engine. Quality control results of sequencing using FASTQC, and raw data processing using HISAT2. Differential analysis of gene expression (DEGs) using edgeR and pathway analysis using DAVID and PANTHER. RESULTS: From the 25,493 genes that experienced a significant change in expression level (P <0.05) from DEG analysis there were 13 genes that play a role in the inflammatory process. Based on DAVID pathway analysis software, there are 8 genes detected based on the KEGG pathway database found in 2 pathways, namely Inflammatory Mediator Regulation of TRP Channels pathway with genes that play HTR2A, NGF, TRPA1, PRKCG, and ADCY8. CXCL9, CXCL10, and CXCL11 genes are found in the Toll-Like Receptor Signaling pathway. Based on PANTHER pathway analysis software, 6 genes were found, namely CXCL10, MYLK2, COL20A1, MYH2, ACTC1, and ALOX15 in the Inflammation Mediated by Chemokine and Cytokine Signaling pathways. Almost all genes found from DEGs are upregulated, except the ALOX15 gene that is downregulated. CONCLUSION: There are 13 genes that play a role in the inflammatory process in Nasopharyngeal Carcinomafrom a sample of the Indonesian population. Genes CXCL9, CXCL10, CXCL11, MYLK2, COL20A1, MYH2, ACTC1, HTR2A, NGF, TRPA1, PRKCG, and ADCY8 have been upregulated and ALOX15 has been downregulated. These genes play a role in the Inflammation Mediated by Chemokine and Cytokine Signaling pathways, Inflammatory Mediator Regulation of TRP Channels, and Toll-Like Receptor Signaling.

Circulation EBV Mir-Bart-7 Relating to Clinical Manifestation in Nasopharyngeal Carcinoma.

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 <0.05). CONCLUSION: Over-expression of circulating EBV miR-BART7 correlated with positive regional lymph nodes reflecting the diagnostic and prognostic values of circulating miR-BART7 for patients with NPC.

Dynamic Changes of Circulating Mir-155 Expression and the Potential Application as a Non-Invasive Biomarker in Breast Cancer.

BACKGROUND: Breast cancer incidence rates have been continuously increasing in majority nations with significant higher portion of cancer-related mortality in low- and middle-income countries. Developing new biomarker is an emerging field in the breast cancer research. Application of a promising minimally invasive biomarker, circulating microRNA, for additional improvement of diagnosis, prognosis, and therapeutic monitoring in breast cancer is not well corroborated. MATERIALS AND METHODS: To uncover the potential use of circulating miR-155 expression as a clinical biomarker in breast cancer, we analyzed 102 breast cancer patients at diagnosis and after treatment as well as 15 healthy women. Total RNA was isolated from patient's plasma and expression of circulating miR-155 was measured with quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression levels of circulating miR-155 were compared according to the effect of treatment, clinicopathological variables, and progression-free survival.  Results: In comparison to the healthy women, expression of circulating miR-155 levels were significantly higher (medians were 18.49±19 and 1.28±0.18, respectively; p<0.0001). The expression levels of miR-155 were significantly diminished after patients completed surgery and chemotherapy (medians were 18.49±19 at diagnosis and 1.32±0.22 after treatment, respectively; p<0.0001). Patients older than 40 years old expressed higher circulating miR-155 than those younger than 40 years-old (medians were 28.92±22 and 4.19±2.49, respectively; p<0.0001). Circulating miR-155 was significantly higher in patients with tumors larger than 5 cm (44.27±2.6 vs 9.17±6.9, p=0.03). MiR-155 expression levels were not significantly different according to various tumor grades, subtypes, and clinical stages. Although longer follow-up is required, progression-free survivals of patients with upregulation of circulating miR-155 were significantly longer (mean survivals were 77 and 65 weeks, Log-rank (Mantel-Cox) test p=0.038). CONCLUSION: Expression of circulating miR-155 expression was significantly elevated in breast cancer patients and was decreased after treatment. Therefore, circulating miR-155 is potentially applicable as diagnostic therapeutic monitoring marker in breast cancer.
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Upregulation of Circulating MiR-21 Expression as a Potential Biomarker for Therapeutic Monitoring and Clinical Outcome in Breast Cancer

Background: Aberrant patterns of microRNA expression have been highlighted as a potential clinical biomarker in breast cancer as the most frequent cancer among women that contributes nearly a quarter of total cancer incidence in 2018. Upregulation of microRNA-21 (miR-21) is associated with adverse clinical outcomes in breast cancer. However, the use of circulating free miR-21 as a non-invasive biomarker for diagnosis and therapeutic monitoring in breast cancer is not well established. We quantified the levels of circulating miR-21 expression and analyzed their correlation with clinicopathological variables and progression-free survival. Materials and Methods: This initial study included a cohort of 102 breast cancer patients of different subtypes and clinicat stages. We also included 15 unrelated healthy women. Venous blood from patients was collected at diagnosis and after treatment of surgery and chemotherapy. MiR-21 expression was quantified from total RNA fraction isolated from patient's plasma. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to analyzed miR-21 expression. Results: Expression of circulating miR-21 was significantly elevated in breast cancer patients compared to healthy women (median miR-21 expression levels were 7.67±2.2 and 1.28±0.16, respectively; p<0.0001). Significant reduction of miR-21 expression was observed in breast cancer patients after completion of surgery and chemotherapy (median miR-21 expression levels were 7.67±2.2 at diagnosis and 2.16±1.28 after treatment, respectively; p<0.0001). MiR-21 expression was higher in breast cancer patients younger than 40-year-old but was not significantly different according to different histopathological grades and clinical stages at diagnosis. Patients with upregulation of circulating miR-21 were associated with poor progression-free survival (median survival 72 vs 86 weeks, respectively; log-rank (Mantel-Cox) test, p=0.049). Conclusion: MiR-21 expression was upregulated in breast cancer patients and might serve as a therapeutic monitoring marker.