as one of the most common intracranial tumors in humans , pituitary adenoma is the third most common primary brain tumor after glioma and meningioma , accounting for about 15~20% of all primary intracranial tumors .
the average incidence of pituitary adenoma is about 7.5~10 per 100 000 population , but has rapidly increased in recent years .
many pituitary tumors are identified by mri or ct when diagnosing nasopharyngeal disease , brain trauma , or other head injury , with an incidence of over 20% .
although it is a benign tumor , some pituitary adenomas showed invasive growth with a malignant tendency , invading peripheral tissues , including bone , dura matter , suprasellar , parasellar , and even cavernous sinus or / and sphenoid sinus , entrenching blood vessels and nerves .
the radical treatment of ipa is extremely difficult because it is hard to completely remove during surgery .
even with post - operative radio-/chemo - therapy , the recurrence rate is still as high as 21~86% .
basic research has been performed to explain the molecular mechanism governing the occurrence and progression of ipa from genetic or protein levels , in an attempt to obtain early diagnosis , prognostic evaluation , and guided treatment .
microrna ( mir ) is a family of endogenous , non - coding , single - stranded rna 21~23 nucleotides in length .
it can regulate gene expression at the post - transcriptional level via inhibiting translation or degrading target mrna .
mir has been found to affect a series of biological processes , including cell proliferation , apoptosis , differentiation , migration , and tumor malignant transformation . the abnormal expression and function of mir
is closely correlated with the occurrence , progression , recurrence , and malignant transformation of ipa .
some scholars used high - throughput microarray technique to detect differential expression of mir across invasive and non - invasive pituitary adenoma .
it has been confirmed that this differential expression of mir is closely correlated with invasive growth of pituitary adenoma .
mir-26a has been demonstrated to be highly expressed in pituitary adenoma , suggesting its possible involvement in tumor occurrence .
pleomorphic adenoma gene 1 ( plag1 ) is a definitive target gene of mir-26a , and is involved in multiple biological effects in regulating cell proliferation , induction of cell apoptosis , and cell cycle arrest . under physiological conditions , plag1 is abundantly expressed in pituitary tissues , but is significantly downregulated in pituitary adenoma .
however , the abnormal expression or function of mir-26a and plag1 related with occurrence of ipa is still unknown . this study aimed to investigate the relationship of pituitary tumor invasion with mir-26a and plag1 .
rna extraction reagent trizol was purchased from invitrogen ( usa ) . reverse transcription and fluorescent quantitative pcr kit were purchased from takara ( china ) .
primers for reverse transcription of mir-26a and pcr were synthesized by ruibo bio ( china ) .
a total of 70 pituitary adenoma patients who received treatment in yantaishan hospital from december 2009 to july 2013 were recruited .
there were 50 males and 20 females , with an age range of 2367 years ( average age , 46.8 years ) .
there were 22 macroadenomas ( tumor size larger than 1 cm ) and 48 microadenomas ( smaller than 1 cm ) cases . in pathological subtyping , there were 14 cases of acth adenomas ( 5 cases of invasion of sphenoid sinus , 6 cases of invasion of the cavernous sinus , 3 cases of invasion of the hypothalamus , 3 cases of < 1-cm microadenoma , 4 cases of 12-cm small adenomas , 5 cases of 23-cm large adenoma , 2 cases of the > 3-cm huge adenoma ; acth of blood > 46 ng / ml , average 156.41 + 45.65 ng / ml ; ufc/24 h > 100 g , average 387.5195.36 g ) , 16 cases of prolactin adenomas ( 7 cases of invasion of sphenoid sinus , 4 cases of invasion of the cavernous sinus , 5 cases of invasion of the hypothalamus ; 4 cases of < 1-cm microadenomas , 8 cases of 13-cm large adenoma , 4 cases of > 3-cm huge adenomas ; prl of serum > 200 ng / ml , average 436.8866.38 ng / ml ) , 15 cases of gh adenoma ( 6 cases of invasion of sphenoid sinus , 4 cases of invasion of cavernous sinus , 5 cases of invasion of the hypothalamus ; 5 cases of < 1-cm micro adenomas , 7 cases of 12-cm small adenomas , 3 cases of 23-cm large adenomas ; serum growth hormone by oral glucose tolerance test > 1 g / l , average 4.520.58 g / l ) and 25 cases of non - functional adenoma ( pituitary tumor was found in sella by ct and mri , no other abnormal increase of pituitary hormone and clinical manifestations were not found except for prl ; serum prolactin levels < 100 ng / ml , average 76.56 + 12.18 ng / ml ) .
the criteria of ipa included : ( 1 ) grade iii~v of hardy - wilson guideline or stage c , d , or e ; ( 2 ) imaging implication before surgery , as shown by destruction of cavernous sinus , parasellar or hypothalamus regions ; ( 3 ) pathological examination indicated tumor infiltration in sellar diaphragm , or sellar floor bones ; and ( 4 ) perforation of inner wall of cavernous sinus during endoscopic surgery .
the criteria of non - invasive pituitary adenoma included focal tumor within sellar region , without compression on peripheral structure as shown by imaging and surgery field .
exclusive criteria were : primary pituitary diagnosis from initially clinical and imaging information , but shown as other tumors such as canalis pharyngeal canal carcinoma , sellar nodular meningioma or rathke s cyst . in the control group ,
a total of 12 normal pituitary tissues were removed from postmortem autopsy of patients without pituitary endocrine disorder .
all sample collections were performed with patient consent , and the study was approved by the ethics committee of yantaishan hospital .
detailed medical history and follow - ups were pursued in all patients via telephone or mail .
post - operative follow - up was terminated on july 2015 , with a range of 165 months ( average , 23.718.6 months ) .
fresh tissue samples collected from the surgery were immediately frozen in liquid nitrogen and were kept at 80c until further use .
uv spectrometry was employed to detect a260/a280 ratio of total rna , which should have the average ratio between 1.8 and 2.0 .
we used 1% agarose gel electrophoresis , which revealed clear and sharp bands of 28s and 28s rna , suggesting high purity , no degradation , and high quality of extracted rna samples .
mir-26a - specific stem - loop primer and random primer were used to synthesize cdna for mir-26a . in a total 20 l reverse transcription system
, there were 2 g total rna , 2 l dntp ( 2.5 mmol / l ) , 4 l rt buffer ( 5x ) , 2 l rt primers ( 1 mol / l ) , 2 l reverse transcriptase , and 0.5 l rnase inhibitor .
the reverse transcription reaction was performed at 16c for 30 min , followed incubation at 42c for 15 min and 85c for 5min .
pcr primers were : mir-26apf : 5-ttgga tccgt cagaa attct ctccc gagg-3 ; mir-26apr : 5-ggtct agatg tgaac tctgg tgttg gtgc-3 ; u6pf : 5-ctcgc ttcgg cagca ca-3 ; u6pr : 5-aacgc ttcac gaatt tgcgt-3 ; plag1pf : 5-atcac ctcca tacac acgac c-3 ; plag1pr : 5-agctt ggtat tgtag ttctt gcc-3 ; -actinpf : 5-gcact cttcc agcct tcc-3 ; and -actinpr : 5-agaaa gggtg taacg caact aag-3. in a total of 10 l reaction system , we added 4.5 l 2x sybr green mixture ; 0.5 l of forward and reverse primers ( 5 m / l each ) , 1 l cdna , and 3.5 l ddh2o .
the reaction conditions were : 95c for 5 min , followed by 40 cycles each of 95c for 15 s and 60c for 1 min .
data were collected on an abi viia7 fluorescent quantitative pcr cycler for comparative ct method ( 2 ) analysis .
the level of microrna and mrna was normalized against u6 or -actin as the internal reference gene .
proteins were then transferred to membrane , which was first blocked in 5% defatted milk powder , and then incubated in rabbit anti - human plag1 antibody at 4c overnight .
unbounded primary antibody was washed , followed by secondary antibody for 60-min incubation at room temperature .
ecl reagent was finally added to develop the membrane , which was exposed and captured for data analysis .
measurement data are presented as mean  standard deviation ( sd ) and enumeration data are presented as percentages . between - group comparison of enumeration data was performed with the chi - square test .
expression level of mir-26a was compared by the mann - whitney u rank - sum test , while the correlation between mir-26a and plag1 was analyzed using the spearman rank - sum test .
the receiver operating characteristic ( roc ) curve was used to evaluate the diagnostic value of mir-26a and plag1 on invasive / non - invasive pituitary adenoma .
the cox regression model was used in multi - variant analysis of related prognostic factors .
rna extraction reagent trizol was purchased from invitrogen ( usa ) . reverse transcription and fluorescent quantitative pcr kit were purchased from takara ( china ) .
primers for reverse transcription of mir-26a and pcr were synthesized by ruibo bio ( china ) .
a total of 70 pituitary adenoma patients who received treatment in yantaishan hospital from december 2009 to july 2013 were recruited .
there were 50 males and 20 females , with an age range of 2367 years ( average age , 46.8 years ) .
there were 22 macroadenomas ( tumor size larger than 1 cm ) and 48 microadenomas ( smaller than 1 cm ) cases . in pathological subtyping , there were 14 cases of acth adenomas ( 5 cases of invasion of sphenoid sinus , 6 cases of invasion of the cavernous sinus , 3 cases of invasion of the hypothalamus , 3 cases of < 1-cm microadenoma , 4 cases of 12-cm small adenomas , 5 cases of 23-cm large adenoma , 2 cases of the > 3-cm huge adenoma ; acth of blood > 46 ng / ml , average 156.41 + 45.65 ng / ml ; ufc/24 h > 100 g , average 387.5195.36 g ) , 16 cases of prolactin adenomas ( 7 cases of invasion of sphenoid sinus , 4 cases of invasion of the cavernous sinus , 5 cases of invasion of the hypothalamus ; 4 cases of < 1-cm microadenomas , 8 cases of 13-cm large adenoma , 4 cases of > 3-cm huge adenomas ; prl of serum > 200 ng / ml , average 436.8866.38 ng / ml ) , 15 cases of gh adenoma ( 6 cases of invasion of sphenoid sinus , 4 cases of invasion of cavernous sinus , 5 cases of invasion of the hypothalamus ; 5 cases of < 1-cm micro adenomas , 7 cases of 12-cm small adenomas , 3 cases of 23-cm large adenomas ; serum growth hormone by oral glucose tolerance test > 1 g / l , average 4.520.58 g / l ) and 25 cases of non - functional adenoma ( pituitary tumor was found in sella by ct and mri , no other abnormal increase of pituitary hormone and clinical manifestations were not found except for prl ; serum prolactin levels < 100 ng / ml , average 76.56 + 12.18 ng / ml ) .
the criteria of ipa included : ( 1 ) grade iii~v of hardy - wilson guideline or stage c , d , or e ; ( 2 ) imaging implication before surgery , as shown by destruction of cavernous sinus , parasellar or hypothalamus regions ; ( 3 ) pathological examination indicated tumor infiltration in sellar diaphragm , or sellar floor bones ; and ( 4 ) perforation of inner wall of cavernous sinus during endoscopic surgery .
the criteria of non - invasive pituitary adenoma included focal tumor within sellar region , without compression on peripheral structure as shown by imaging and surgery field .
exclusive criteria were : primary pituitary diagnosis from initially clinical and imaging information , but shown as other tumors such as canalis pharyngeal canal carcinoma , sellar nodular meningioma or rathke s cyst . in the control group ,
a total of 12 normal pituitary tissues were removed from postmortem autopsy of patients without pituitary endocrine disorder .
all sample collections were performed with patient consent , and the study was approved by the ethics committee of yantaishan hospital .
detailed medical history and follow - ups were pursued in all patients via telephone or mail .
post - operative follow - up was terminated on july 2015 , with a range of 165 months ( average , 23.718.6 months ) .
fresh tissue samples collected from the surgery were immediately frozen in liquid nitrogen and were kept at 80c until further use .
uv spectrometry was employed to detect a260/a280 ratio of total rna , which should have the average ratio between 1.8 and 2.0 .
we used 1% agarose gel electrophoresis , which revealed clear and sharp bands of 28s and 28s rna , suggesting high purity , no degradation , and high quality of extracted rna samples .
mir-26a - specific stem - loop primer and random primer were used to synthesize cdna for mir-26a . in a total 20 l reverse transcription system ,
there were 2 g total rna , 2 l dntp ( 2.5 mmol / l ) , 4 l rt buffer ( 5x ) , 2 l rt primers ( 1 mol / l ) , 2 l reverse transcriptase , and 0.5 l rnase inhibitor .
the reverse transcription reaction was performed at 16c for 30 min , followed incubation at 42c for 15 min and 85c for 5min .
products were stored at 20c . using cdna as the template , pcr amplification was performed with taqdna polymerase .
pcr primers were : mir-26apf : 5-ttgga tccgt cagaa attct ctccc gagg-3 ; mir-26apr : 5-ggtct agatg tgaac tctgg tgttg gtgc-3 ; u6pf : 5-ctcgc ttcgg cagca ca-3 ; u6pr : 5-aacgc ttcac gaatt tgcgt-3 ; plag1pf : 5-atcac ctcca tacac acgac c-3 ; plag1pr : 5-agctt ggtat tgtag ttctt gcc-3 ; -actinpf : 5-gcact cttcc agcct tcc-3 ; and -actinpr : 5-agaaa gggtg taacg caact aag-3. in a total of 10 l reaction system , we added 4.5 l 2x sybr green mixture ; 0.5 l of forward and reverse primers ( 5 m / l each ) , 1 l cdna , and 3.5 l ddh2o .
the reaction conditions were : 95c for 5 min , followed by 40 cycles each of 95c for 15 s and 60c for 1 min .
data were collected on an abi viia7 fluorescent quantitative pcr cycler for comparative ct method ( 2 ) analysis .
the level of microrna and mrna was normalized against u6 or -actin as the internal reference gene .
proteins were then transferred to membrane , which was first blocked in 5% defatted milk powder , and then incubated in rabbit anti - human plag1 antibody at 4c overnight .
unbounded primary antibody was washed , followed by secondary antibody for 60-min incubation at room temperature .
ecl reagent was finally added to develop the membrane , which was exposed and captured for data analysis .
measurement data are presented as mean  standard deviation ( sd ) and enumeration data are presented as percentages . between - group comparison of enumeration data
expression level of mir-26a was compared by the mann - whitney u rank - sum test , while the correlation between mir-26a and plag1 was analyzed using the spearman rank - sum test .
the receiver operating characteristic ( roc ) curve was used to evaluate the diagnostic value of mir-26a and plag1 on invasive / non - invasive pituitary adenoma .
the cox regression model was used in multi - variant analysis of related prognostic factors .
qrt - pcr results showed that , compared to normal pituitary tissues , mir-26a was significantly upregulated in pituitary tumor tissues ( figure 1a ) , while plag1 mrna level was remarkably decreased ( figure 1b ) .
ipa tissues had significantly higher mir-26a and lower plag1 mrna compared to non - invasive pituitary tumor tissues .
western blotting obtained consistent results , as normal pituitary tissues had higher plag1 protein levels compared to those in non - invasive pituitary adenoma tissues , which again had higher plag1 protein compared to ipa tissues ( figure 1c ) .
further correlation analysis revealed a significant positive correlation between plag1 mrna and protein expression levels ( r=0.635 , p=0.019 ) and a significant negative correlation between mir-26a and plag1 mrna expression level ( r=0.791 , p=0.021 ) , supporting the targeted regulation of mir-26a on plag1 . using median values of mir-26a or plag1 mrna expression levels of all patients as boundary lines , we further divided all patients into high-/low - expression subgroups .
there was a significant difference in mir-26a or plag1 expression levels between non - ipa and ipa patients ( p=0.004 and 0.006 , respectively ) .
the ratio of mir-26a high - expression in ipa patients was significantly higher than that in non - ipa patients ( p=0.004 ) , and higher plag1 low - expression population ratios ( p=0.006 ) .
we did not observe a significant relationship between mir-26a or plag1 expression levels and patient age , sex , tumor size , or adenoma subtypes ( p>0.05 in all cases , table 1 ) .
using hardy classification as the criterion standard , we divided pituitary adenomas into invasive and non - invasive types . by constructing an roc , we calculated the classification accuracy of mir-26a / plag1 in differentiating invasive from non - invasive pituitary adenomas .
the results showed certain differential diagnostic values of those 2 genes , as mir-26a had auc values of 0.889 for differential diagnosis between invasive and non - invasive pituitary adenoma , higher than that of plag1 ( auc=0.818 , figure 2 ) .
those patients with mir-26a high - expression had a significantly sharper survival curve than those with lower mir-26a expressions .
in contrast , plag1 low - expression patients had a significantly sharper survival curve than those with higher plag1 expression ( figure 3a , 3b ) .
log - rank test results showed significantly lower survival rate of mir-26a high - expression patients than those with lower mir-26a expression ( =7.393 , p=0.007 ) , while patients with lower plag1 expression had significantly lower survival rates than those with higher plag1 expression ( =9.475 , p=0.002 ) .
for patients with both mir-26a high - expression and plag1 low - expression , the prognosis was remarkably worse than in other patients ( =11.400 , p<0.001 , figure 3c ) .
we performed a cox regression analysis including age , sex , tumor size , invasiveness , adenoma type , mir-26a expression level , and plag1 expression level . using the maximal likelihood estimation biased forward selection method , we designated tumor invasiveness , mir-26a expression level , and plag1 expression level as independent risk factors affecting the survival of pituitary adenoma patients ( table 2 ) . invasiveness increased the death risk of pituitary adenoma patients by 0.899-fold ( p=0.009 ) . mir-26a
high - expression increased the death risk by 1.833-fold compared to that in low - expression patients ( p=0.022 ) . on the contrary , elevated plag1 expression protected patients , reducing death risk by 39.7% ( p=0.018 ) .
qrt - pcr results showed that , compared to normal pituitary tissues , mir-26a was significantly upregulated in pituitary tumor tissues ( figure 1a ) , while plag1 mrna level was remarkably decreased ( figure 1b ) .
ipa tissues had significantly higher mir-26a and lower plag1 mrna compared to non - invasive pituitary tumor tissues .
western blotting obtained consistent results , as normal pituitary tissues had higher plag1 protein levels compared to those in non - invasive pituitary adenoma tissues , which again had higher plag1 protein compared to ipa tissues ( figure 1c ) .
further correlation analysis revealed a significant positive correlation between plag1 mrna and protein expression levels ( r=0.635 , p=0.019 ) and a significant negative correlation between mir-26a and plag1 mrna expression level ( r=0.791 , p=0.021 ) , supporting the targeted regulation of mir-26a on plag1 .
using median values of mir-26a or plag1 mrna expression levels of all patients as boundary lines , we further divided all patients into high-/low - expression subgroups . there was a significant difference in mir-26a or plag1 expression levels between non - ipa and ipa patients ( p=0.004 and 0.006 , respectively ) .
the ratio of mir-26a high - expression in ipa patients was significantly higher than that in non - ipa patients ( p=0.004 ) , and higher plag1 low - expression population ratios ( p=0.006 ) .
we did not observe a significant relationship between mir-26a or plag1 expression levels and patient age , sex , tumor size , or adenoma subtypes ( p>0.05 in all cases , table 1 ) .
using hardy classification as the criterion standard , we divided pituitary adenomas into invasive and non - invasive types . by constructing an roc , we calculated the classification accuracy of mir-26a / plag1 in differentiating invasive from non - invasive pituitary adenomas .
the results showed certain differential diagnostic values of those 2 genes , as mir-26a had auc values of 0.889 for differential diagnosis between invasive and non - invasive pituitary adenoma , higher than that of plag1 ( auc=0.818 , figure 2 ) .
those patients with mir-26a high - expression had a significantly sharper survival curve than those with lower mir-26a expressions .
in contrast , plag1 low - expression patients had a significantly sharper survival curve than those with higher plag1 expression ( figure 3a , 3b ) .
log - rank test results showed significantly lower survival rate of mir-26a high - expression patients than those with lower mir-26a expression ( =7.393 , p=0.007 ) , while patients with lower plag1 expression had significantly lower survival rates than those with higher plag1 expression ( =9.475 , p=0.002 ) .
for patients with both mir-26a high - expression and plag1 low - expression , the prognosis was remarkably worse than in other patients ( =11.400 , p<0.001 , figure 3c ) .
we performed a cox regression analysis including age , sex , tumor size , invasiveness , adenoma type , mir-26a expression level , and plag1 expression level . using the maximal likelihood estimation biased forward selection method , we designated tumor invasiveness , mir-26a expression level , and plag1 expression level as independent risk factors affecting the survival of pituitary adenoma patients ( table 2 ) . invasiveness increased the death risk of pituitary adenoma patients by 0.899-fold ( p=0.009 ) . mir-26a
high - expression increased the death risk by 1.833-fold compared to that in low - expression patients ( p=0.022 ) . on the contrary , elevated plag1 expression protected patients , reducing death risk by 39.7% ( p=0.018 ) .
pituitary tumors are monoclonal tumors originating from residual epithelial cells of anterior / posterior pituitary and cranial - pharyngeal tissues , and account for about 10~15% of all intracranial tumors .
clinically , however , about one - third of all pituitary tumors showed invasive growth , which is a biological behavioral of malignant tumors .
such pituitary adenomas often show aggressive growth toward peripheral tissues / nerves , and thus are termed invasive pituitary tumors .
invasive pituitary tumors have rapid progression , are difficult to completely removal during surgery , and have higher short - term recurrence rate after surgery , making clinical treatment a major challenge .
the invasive growth of pituitary tumor is one important factor causing the recurrence of disease , further affecting clinical treatment efficacy and prognosis .
recent studies have focused on the mechanism directing pituitary tumor proliferation , activation of oncogenes , and inactivation of tumor suppression genes , but the biological mechanism affecting tumor invasion / migration is still not fully understood .
for instance , ccnd1 gene , a proto - oncogene , plays a role in pituitary tumorigenesis and invasiveness and its polymorphism in patients with different types of sporadic pituitary adenomas were determined .
hypoxia can increase the expression of ddr1 , a newly discovered kind of tyrosine kinase receptor on the cell surface , which in turn promotes pituitary adenoma cell proliferation and invasion .
in particular , a recent finding showed that the expression of cold - inducible rna - binding protein ( cirp ) in pituitary adenomas is closely related with tumor proliferation and invasion , and its significantly elevated expression level indicates post - operative recurrence .
therefore , understanding the invasion / migration mechanism and identifying the specific molecular marker for acquiring invasion / migration property are of critical importance for early diagnosis of pituitary tumors , broadening clinical treatment strategy , improving treatment efficacy , and guiding individualized treatment . with the advancement of molecular biology ,
the pathogenesis mechanism of ipa has been illustrated from the molecular level by some scholars .
many signal molecules have been discovered during the modulation of cell proliferation , cycle , apoptosis , and differentiation , especially the critical role during tumor cell occurrence and invasion / migration , thus providing new evidence depicting the tumor invasion mechanism and advancing targeted treatment .
microrna has been found to participate in occurrence , progression , invasion , and metastasis of pituitary adenoma via degrading target gene mrna or inhibiting their translation .
some researchers utilized high - throughput microarray to reveal differential mir expressions between invasive / non - invasive pituitary adenoma . moreover ,
such differential expression of mir has been found to be closely correlated with invasive growth of pituitary adenomas .
bottoni et al . utilized microarray analysis to screen the differential expression of mir between pituitary adenoma and normal pituitary tissues , and found the overexpression of mir-26a in pituitary adenoma , indicating its possible involvement in occurrence of pituitary adenoma .
pleomorphic adenoma gene 1 ( plag1 ) is located in human chromosome 8q12 , with 7313bp full - length cdna .
plag1 encodes 1 nuclear protein , which belongs to the zinc - finger protein family . with transcription factor activity
a study has confirmed plag1 as a target gene for mir-26a , which regulates plag1 gene expression . under physiological conditions ,
plag1 is abundantly expressed in pituitary tissues , but is significantly downregulated in pituitary adenoma .
the correlation between mir-26a / plag1 expression or function abnormality and the occurrence of pituitary adenoma is still unknown , as its relationship with the invasiveness of pituitary adenoma . the results of the present study show that , compared to normal pituitary tissues , pituitary adenoma tissues have significantly elevated mir-26a expression , indicating its potential involvement with pituitary adenoma occurrence .
gentilin et al . found significantly elevated mir-26a expression in the acth - type pituitary adenoma cell line att20/d16v - f2 .
such upregulated mir-26a directly suppressed the expression of prkcd ( protein kinase c ) , and facilitated the expression of cell cycle modulatory protein cyclin e and cyclin a , thus shortening the g1 phase and accelerating the cell cycle , indicating the facilitating role of mir-26a in pituitary adenoma , consistent with our observations .
they also revealed more abundantly expressed mir-26a in ipa tissues compared to non - ipa tissues , suggesting the correlation between mir-26a and invasiveness of pituitary adenoma , in addition to tumor occurrence .
the study revealed lower expression level of plag1 in pituitary adenoma tissues compared to that in normal pituitary tissues , especially in ipa tissues , suggesting the correlation of plag1 low - expression in occurrence and invasiveness of pituitary adenoma .
however , no statistical significance was found in the proportion of high and low expressions of mir-26a and plag1 within different functional types of adenomas .
found the physiological expression of plag1 protein in pituitary tissues and several other brain regions .
ttt / gf significantly enhanced cell proliferation ability , suggesting the role of plag1 in anti - pituitary tumor proliferation .
moreover , plag1 functions as a tumor suppressor gene in pituitary adenoma via facilitating cell apoptosis , inducing g1 phase arrest .
ki-67 is a critical marker for differential evaluation of mitotic index between non - invasive and invasive pituitary adenoma , and is also an important marker determining the invasiveness of pituitary adenoma .
thapar et al . found significantly higher mitotic index of ipa cells than those in non - invasive pituitary adenoma , suggesting that more active mitosis and uncontrolled tumor cell growth are critical factors for acquiring invasiveness by pituitary adenoma .
therefore , the study showed that elevated mir-26a expression can affect the invasiveness of pituitary adenoma via targeted inhibition on plag1 expression , and antagonizing the tumor suppression effect of plag1 against pituitary adenoma cells .
moreover , they analyzed the correlation between mir-26a / plag1 expression and clinical features of patients , and found the relationship between expression levels and tumor invasiveness .
the study found higher diagnostic values of mir-26a and plag1 in differentiating invasive and non - invasive pituitary adenoma , indicating that the utilization of mir-26a and plag1 expression levels could effectively differentiate and evaluate the invasiveness of pituitary adenoma . compared to classical pre - operative imaging examination , endoscopy during the surgery , and post - operative pathological examination ,
the early clarification of tumor invasiveness by measuring mir-26a and plag1 expression levels is critical importance for early diagnosis and treatment of disease .
the study found that mir-26a and plag1 expression levels are independent risk factors governing patient prognosis , and significantly affect patient s survival and prognosis .
in summary , mir-26a plays a critical role during the occurrence and progression of pituitary adenoma , and may facilitate tumor occurrence and the acquisition of invasiveness , probably via inhibiting plag1 expression .
our study provides new concepts for illustrating the mechanism of the invasiveness of pituitary adenoma , and provides novel methods and approaches for biological therapy against pituitary adenoma , justifying further exploration .
 backgroundalthough pituitary adenoma is a malignant tumor , it can present as invasive growth in some cases . 
 microrna ( mir)-26a has been found to be abnormally highly expressed in pituitary adenoma , indicating possible involvement in pathogenesis . as a known target gene of mir-26a , plag1 has abnormally low expression in pituitary adenoma . 
 the correlation between mir-26a or plag1 expressional abnormality and occurrence of pituitary adenoma is still unknown , as is its association with invasiveness of pituitary adenoma.material/methodspituitary adenoma tissues , including both invasive and non - invasive subtypes , were collected from our neurosurgery department , in parallel with normal pituitary tissues from postmortem autopsy . 
 qrt - pcr was used to detect mrna expression of mir-26a and plag1 , while western blotting was used to test plag1 protein expression . 
 the correlation between mir-26a and plag1 , and with pathological features , were analyzed . 
 roc analysis revealed the utility of mir-26a and plag1 in differential diagnosis of invasive / non - invasive pituitary tumors and in analyzing their effects on patient prognosis.resultsmir-26a was remarkably upregulated in pituitary tumors , while plag1 was downregulated , especially in invasive pituitary tumors . mir-26a and plag1 had higher diagnostic values for differentiating between invasive and non - invasive pituitary tumors ( auc=0.889 and 0.818 , respectively ) . those patients with 
 mir-26 overexpression and plag1 downregulation had unfavorable prognosis . mir-26 and plag1 are independent factors affecting patient diagnosis.conclusionsmir-26a can facilitate occurrence of pituitary tumor and invasiveness , probably via inhibiting plag1 expression . 
Background
Material and Methods
Reagent and materials
Clinical information
qRT-PCR for gene expression
Western blotting
Statistical analysis
Results
MiR-26a and PLAG1 mRNA expression in pituitary adenoma patients
Correlation between mir-26a, PLAG1 expression, and clinical features of patients
Differential diagnosis of pituitary adenoma by miR-26a and PLAG1
Prognosis of miR-26a/PLAG1 expression patients
Independent risk factors affecting patient survival by Cox regression analysis
Discussion
Conclusions