Human of miRNAs could be act in the

Human gastric cancer (GC) is still a third cause of cancer related mortality
in developed countries (1). As estimated GC is fifth common cancer worldwide
and responsible for 984,000 new cases and 841,000 deaths were accounted in
2013(2). Although managements and conventional strategies are applied for the
treatment of gastric cancer, satisfactory methods has not yet found. Therefore,
more investigation to finding some new predictive and diagnostic markers are
warranted, which could be helpful in finding new therapeutic strategies to
improving prognosis. Hence, studies about microRNAs might shed light on this
problem (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 to
negatively regulate gene expression via either translational repression or mRNA
(4). It has been reported that altered expression of miRs is  associated with human diseases, particularly
cancers (5) that altered expression patterns of miRNAs could be act in the
oncogenic or tumor suppressor pathways 14-16. Furthermore, altered miRNA
expression 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 can
be helpful to cancer detection and treatment.

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miR-7 is an evolutionarily conserved miRNA. In
humans, 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-7
sequence (20). Several studies reported the tumor suppressive role/down
regulation of mir-7 in pancreatic carcinoma (20), non-small-cell lung cancer(21) and
other cancer cell and tissues(22-24). About a pathway or sth that is common in this cancer
related miR.

In the present study, we
investigated the expression level of miR-7 in gastric dysplasia, GC and
adjacent non-tumor tissue. Such
investigations could help us to develop strategies for future prevention, diagnosis
and treatment of gastric cancer.

 

Materials and Method

tissue samples

A total of 115 formalin-fixed paraffin-embedded
(FFPE) tissue samples include 40 GC, 31 adjacent non-tumor tissue and 44 gastric
dysplasia samples, were obtained from SBMU Endocrine Research Center (ERC)
tissue bank (Tehran, Iran) between 2015 to 2017. The study procedure was approved by the organization
ethical committee (IR.SBMU.MSP.REC.1396.415). Prepared hematoxylin and eosin
stained slide of every sample was reviewed by a member of the Institute of
Pathology at the of Shahid beheshti medical university. The area of interest
was marked and the slide is used to guide for scraping the unstained sections
for tumor, adjacent non-tumor and dysplasia tissue. All samples had a clear
histological diagnosis consistent with gastric dysplasia and gastric cancer
based on the pathology report. All data, including age, gender, lymph node
metastasis (LNM), and histological grade were obtained from clinical and
pathologic records. Patients that received neoadjuvant chemotherapy or
radiotherapy were excluded from the study.

RNA isolation and reverse transcription (cDNA synthesis)

Fifteen-µm microtome-cut sections from each sample was processed and
placed in 1.5-mL nuclease-free microcentrifuge tube. Thereafter, total RNA
extraction were performed by Qiagen miRNeasy FFPE kit (Qiagen, Hilden, Germany)
according to the manufacturer’s protocol and extracted RNA subjected to DNase I treatment to
remove any genomic DNA contamination. The purity
and concentration of extracted RNAs were assessed using
the Nanodrop ND-2000c spectrophotometer (Thermo Scientific).

we used poly(A)-tailed universal reverse transcription method as
described elsewhere (12)
For cDNA synthesis. In brief, 1?g
total RNA containing miRNA was polyadenylated by  E.Coli poly(A)  polymerase (NEB)  at  37°C  for  30
min . Then after, by using the PrimeScript™ RT reagent Kit (TaKaRa, Japan) and
RT primer (Table 1), poly-adenylated RNA was reverse-transcribed. The cDNA
synthesis reaction (10uL) contained polyadenylated RNA(100 ng), 5X PrimeScript
Buffer (2 µL), PrimeScript RT  Enzyme Mix
I (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 for
5 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 with
SYBR Premix Ex TaqTM II (TAKARA) in 15 µl final volumes with using
1.5 µl template  cDNA  mixed 
with  7.5 µl  2× 
SYBR Green PCR master mix and 0.75 µl of each forward and reverse
primers (Table 1). We performed PCR in duplicate according to the
standard 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 verify
ampli?cation speci?city. The change in amplification was normalized to the expression of the
U6 snRNA, as internal control as it did not differ significantly between tumor
and non-tumor tissue in our study population (P-value > 0.05) (Fig.
??). The fold change in expression was calculated using 2-??Ct
method.

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 tissue
showed no significant differences. These values represent the Ct mean of normal
versus tumor groups (P>0.05)

Statistical analysis

All statistical analyses were performed using the SPSS 11.0
software (SPSS Inc.,Chicago, IL, USA). Differences
of miRs expression in the three groups (GC, GD and normal adjacent) were assessed by ordinary one-way
ANOVA analysis. The Student’s unpaired t-test was used to determine the associations of miRs expression and clinicopathological
features. Data are presented as mean ± SEM. P-value ? 0.05 was in all
cases considered statistically significant.

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