Type: Research Essays
Sample donated: Elsie Kim
Last updated: March 22, 2019
Human gastric cancer (GC) is still a third cause of cancer related mortalityin developed countries (1). As estimated GC is fifth common cancer worldwideand responsible for 984,000 new cases and 841,000 deaths were accounted in2013(2). Although managements and conventional strategies are applied for thetreatment of gastric cancer, satisfactory methods has not yet found. Therefore,more investigation to finding some new predictive and diagnostic markers arewarranted, which could be helpful in finding new therapeutic strategies toimproving prognosis. Hence, studies about microRNAs might shed light on thisproblem (3).
MicroRNAs (miRs) form an abundant class of short regulatory (?21-22 nt)non-coding RNAs widely expressed in living organisms with the unique ability tonegatively regulate gene expression via either translational repression or mRNA(4). It has been reported that altered expression of miRs is associated with human diseases, particularlycancers (5) that altered expression patterns of miRNAs could be act in theoncogenic or tumor suppressor pathways 14-16. Furthermore, altered miRNAexpression has been reported in various cancers including lung cancers 8, bladder cancer 9, breast cancer 10, cervical cancer 11,colorectal cancer 12 and also gastric cancer 17-19. so targeting miRNAs canbe helpful to cancer detection and treatment.miR-7 is an evolutionarily conserved miRNA. Inhumans, three miR-7 genes (MIR7-1,2,3) are located on chromosomes 9,15 and 19,respectively.
All these three genes giving rise to the same mature miR-7sequence (20). Several studies reported the tumor suppressive role/downregulation of mir-7 in pancreatic carcinoma (20), non-small-cell lung cancer(21) andother cancer cell and tissues(22-24). About a pathway or sth that is common in this cancerrelated miR.In the present study, weinvestigated the expression level of miR-7 in gastric dysplasia, GC andadjacent non-tumor tissue. Suchinvestigations could help us to develop strategies for future prevention, diagnosisand treatment of gastric cancer. Materials and Methodtissue samplesA total of 115 formalin-fixed paraffin-embedded(FFPE) tissue samples include 40 GC, 31 adjacent non-tumor tissue and 44 gastricdysplasia samples, were obtained from SBMU Endocrine Research Center (ERC)tissue bank (Tehran, Iran) between 2015 to 2017. The study procedure was approved by the organizationethical committee (IR.SBMU.
MSP.REC.1396.415). Prepared hematoxylin and eosinstained slide of every sample was reviewed by a member of the Institute ofPathology at the of Shahid beheshti medical university. The area of interestwas marked and the slide is used to guide for scraping the unstained sectionsfor tumor, adjacent non-tumor and dysplasia tissue.
All samples had a clearhistological diagnosis consistent with gastric dysplasia and gastric cancerbased on the pathology report. All data, including age, gender, lymph nodemetastasis (LNM), and histological grade were obtained from clinical andpathologic records. Patients that received neoadjuvant chemotherapy orradiotherapy were excluded from the study.
RNA isolation and reverse transcription (cDNA synthesis)Fifteen-µm microtome-cut sections from each sample was processed andplaced in 1.5-mL nuclease-free microcentrifuge tube. Thereafter, total RNAextraction were performed by Qiagen miRNeasy FFPE kit (Qiagen, Hilden, Germany)according to the manufacturer’s protocol and extracted RNA subjected to DNase I treatment toremove any genomic DNA contamination. The purityand concentration of extracted RNAs were assessed usingthe Nanodrop ND-2000c spectrophotometer (Thermo Scientific).
we used poly(A)-tailed universal reverse transcription method asdescribed elsewhere (12)For cDNA synthesis. In brief, 1?gtotal RNA containing miRNA was polyadenylated by E.Coli poly(A) polymerase (NEB) at 37°C for 30min . Then after, by using the PrimeScript™ RT reagent Kit (TaKaRa, Japan) andRT primer (Table 1), poly-adenylated RNA was reverse-transcribed. The cDNAsynthesis reaction (10uL) contained polyadenylated RNA(100 ng), 5X PrimeScriptBuffer (2 µL), PrimeScript RT Enzyme MixI (0.5 µL), RT primer mixture (1 µL equivalent 100 pmol), and RNase-free water.Then, the total reaction mixture was incubated at 50°C for 15 min and 85°C for5 sec.
Amplifcation of the human miR?7 Real-time RT–PCR was performed using SYBR Premix Ex TaqII (Takara,Tokyo, Japan) and Rotor Gene 5-plex HRM (Qiagene Technologies). Real-time PCR was performed withSYBR Premix Ex TaqTM II (TAKARA) in 15 µl final volumes with using1.5 µl template cDNA mixed with 7.5 µl 2× SYBR Green PCR master mix and 0.
75 µl of each forward and reverseprimers (Table 1). We performed PCR in duplicate according to thestandard program on Rotor-Gene Q instrument (QIAGEN): 30 second at 95°C,followed by two cycles of amplification (10 sec at 95°C, 30 sec min at 60°C)and ?nally a dissociation curve step (ramp from 70 to 90°C) to verifyampli?cation speci?city. The change in amplification was normalized to the expression of theU6 snRNA, as internal control as it did not differ significantly between tumorand non-tumor tissue in our study population (P-value > 0.05) (Fig.
??). The fold change in expression was calculated using 2-??Ctmethod.Table 1.
Sequences of primers used in this study Primers Sequence RT Primer 5?- GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTTTVN-3? (V= A, G, or C; N= A, G, C, or T.) Universal Primer 5?- GCGAGCACAGAATTAATACGACTC-3? miR-7 5?- GGCGTCTTTGGTTATCTAGCTGTAT-3? U6 5′-CGCAAGGATGACACGCAAATTC-3? Fig. ?? The expression level of U6 in GC compared to adjacent normal tissueshowed no significant differences. These values represent the Ct mean of normalversus tumor groups (P>0.05)Statistical analysisAll statistical analyses were performed using the SPSS 11.0software (SPSS Inc.
,Chicago, IL, USA). Differencesof miRs expression in the three groups (GC, GD and normal adjacent) were assessed by ordinary one-wayANOVA analysis. The Student’s unpaired t-test was used to determine the associations of miRs expression and clinicopathologicalfeatures. Data are presented as mean ± SEM. P-value ? 0.05 was in allcases considered statistically significant.