| Abstract |
Although it is well established that ras genes contribute to tumourigenesis
either through the accumulation of mutations or by aberrant expression in a wide
range of human cancers, little is known regarding their involvement in human nasal
polyps (NPs). Moreover, little is known about the implication of BRAF and RKIP
expression, or about the incidence of BRAF mutations, as well as about the presence of
viral DNA in the formation of nasal polyps.
In the present study, the occurrence of mutations in codons 11, 12 and 13 of
the ras family genes was examined by PCR/RFLP and direct sequencing in 23 human
NPs and their adjacent turbinates [Control Inferior Turbinates (CIT) and Control Middle
Turbinates (CMT)], as well as in turbinates from 13 control subjects (CIT and CMT).
Moreover, the expression pattern of ras mRNA levels was assessed in NP specimens
and compared to adjacent and control tissues. Regarding the second part of this study,
we determined the expression levels of the genes BRAF and RKIP, and inspected the
frequency of BRAF mutations in exons 11, 14 and 15 in the nasal polyps and their
adjacent and control turbinates. We analysed 24 NPs and their adjacent turbinates
(CIT and CMT), as well as 14 CITs and 14 CMTs. The expression pattern of BRAF and
RKIP was assessed with real-time RT-PCR. A real-time allele-specific PCR method, in
combination with direct sequencing, was performed in order to inspect the frequency
of the V600E mutation in exon 15, and to examine mutation status within exons 11
and 14. The presence of amplifiable DNA was confirmed with β2-microglobulin primers,
and the HPV, EBV and HSV viral DNA detection was performed with specific primer
sets, using PCR.
K-ras codon 11 and 12 mutations were detected in 17 and 35% of NPs,
respectively, and were found in the adjacent inferior turbinate (AIT) (22 and 16%,
respectively) and adjacent middle turbinates (AMT) (16 and 26%, respectively). K- and
H-ras expression levels were elevated, whereas N-ras mRNA levels were lower in NPs
and adjacent turbinates as compared to the control tissues. K-ras mRNA levels were
up-regulated in advanced-stage polyps (P=0.037), while N-ras levels were found
elevated in small polyps (P=0.046). Statistically significant negative correlations
between K- and N-ras expression profiles arose in NPs and AITs (P=0.009 and 0.003,
respectively). Regarding the second part of our study, our results were as follows: The
control mucosae presented significantly higher mRNA levels for both genes, compared
to the NP and the AIT–AMT. Moreover, in NP, AIT and AMT, RKIP was found to
present higher mRNA levels, in relation to the equivalent values of the BRAF gene (P =
0.003 in NP; P < 0.001 both in AIT and AMT). No mutation was detected in exon 14,
whereas a silent mutation (A1380G, G460G) was noted for one NP sample in exon 11.
Another NP sample was found to carry two mutations, one T1799A (V600E) and one
A1801G (K601E). A significant co-expression of the two genes was noted in NP (P =
0.012) and AIT (P = 0.019). Regarding the viral DNA detection, three NP samples
(3/23, 13%) and one sample from each adjacent turbinate [AIT, 123 (4%); AMT, 1/12
(4%)] were found to be positive for the HPV virus, yet no oncogenic type (HPV-11, 16,
-18, -33) was recognized. Eight NP samples (8/23, 35%) were EBV positive, and two
NP samples (2/23, 8%) were HSV positive, respectively. No adjacent turbinate was
positive for the presence of neither of these two herpes viruses. Moreover, al the
control turbinates were also found to be negative for the presence of the HPV, EBV
and HSV viruses.
This, to our knowledge, is the first report on ras mutations and expression
analysis in NPs. Our findings suggest a potential key role for activated members of ras
family genes in terms of their contribution to the development of NPs as well as to the
hypertrophy of adjacent turbinates. The results of the expression levels of RKIP and
BRAF, reflect the strong connection between the two genes. RKIP could play an
important role in the down-regulation of wild-type BRAF, serving thus as an
endogenous inhibitor of the MAPK pathway in nasal polyps and their adjacent turbinate
mucosa. The results upon the presence of viral DNA in the nasal polyps, confirm the
hypothesis that HPV is not frequently associated with sinunasal polyposis, and it is
correlated to a lesser extent to infectious mucosal lesions, than to proliferative lesions.
Regarding EBV, the percentage of infection shows that nasal mucosa could be one of
the sites of persistence of the virus, and there’s a possibility that it is responsible for
presence of viral-associated nasal tumors, whereas, HSV does not seem to be
implicated in the development of nasal polyps.
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