osteosarcoma is a common primary malignant bone tumor with complicated pathogenesis and frequent distal metastasis , thus causing higher mortality rates , especially in the teenagers .
epidemiological surveys revealed that nearly 40% of osteosarcoma patients died from misdiagnosis and/or early tumor metastasis , especially toward the lung tissue [ 24 ] .
therefore , it is of critical importance to find a simple and effective biological marker for osteosarcoma , in order to make early diagnosis .
current studies have revealed the role of gene mutation in the proliferation of tumor cells .
cell apoptosis - related genes , including vascular epithelial growth factor ( vegf ) and microrna ( mir)-96 , were found to be closely correlated with the occurrence and prognosis of osteosarcoma .
p15 , a novel tumor - suppressor gene , has been reported to play an important regulatory role in the progression of various malignant tumors , such as pulmonary cancer , nasopharynx cancer , and kidney cancer .
the fragment loss , point mutation , or methylation of p15 gene may all lead to its abnormal tumor - suppressing function , thus facilitating the malignancy and metastasis of tumor cells .
previous studies have been performed on the pathogenesis and related mechanisms of pulmonary metastasis of osteosarcoma , but the involvement of p15 gene has not been reported . as it is of critical importance for early diagnosis of osteosarcoma to discover a simple but effective biological tumor marker , this study aimed to describe the mechanism of p15 gene mutation in the occurrence and lung metastasis of osteosarcoma in a rat model , as previously described .
this study may provide further evidence for developing methods in clinical diagnosis of malignant osteosarcoma .
a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) .
the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group .
rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university .
after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues .
the tumor volume was approximated as : ( short diameter  long diameter)/2 , as reported elsewhere .
the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration .
we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature .
pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody .
after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times ,
3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min .
dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting .
sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields
we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) .
peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above .
after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents .
total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) ,
pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) .
the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min .
agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) .
spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison
a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) .
the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group .
rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university .
after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues .
morphological changes were observed , along with the measurement of tumor size . the tumor volume was approximated as : ( short diameter  long diameter)/2 , as reported elsewhere .
the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration .
we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature .
pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody .
after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times , 3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min .
dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting .
sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields
we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) .
peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above .
after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents .
total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) ,
pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) .
the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min .
agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) .
spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison
one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites .
as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) .
abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week
, the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues .
on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly .
a longitudinal dissection of the tumor revealed white and hard tissues with centralized necrosis . before tumor cell inoculation
after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time .
on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) .
in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points
further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) .
one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites .
as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) .
abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week
, the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues .
on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly .
after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time .
on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) .
in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points
further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) .
due to its unfavorable prognosis , early diagnosis and treatment is of critical importance for preventing metastasis of osteosarcoma lesions .
the clinical features of osteosarcoma mainly include bone pain , hypercalcinemia , and osteolysis metastasis .
the complication of other organ / tissues , such as the lung , may indicate the metastasis of tumors .
the proliferation of primary tumor lesions causes the detachment of tumor cells , which penetrate vessel walls to enter the general lymph or blood circulation system for the invasion of distal tissues , where tumor  seeds 
investigation of the mechanisms underlying the progression and metastasis of osteosarcoma is a popular research topic .
recent studies revealed complicated genetic processes regulating the tumor metastasis , as a  hot - spot  located in chromosome 9p21 - 22 region affects gene fragment loss or transcriptional abnormalities in various malignant tumors [ 1719 ] .
p15 gene , as a representative gene in this region , has been shown to have mutants and expression down - regulation in kidney and bladder cancer . in approximately 23% of cancer cells of
it has been indicated that single - base mutation occurs in 67 , 119 , and 135 codons of p15 gene . due to the methylation of p15 gene ,
hematopoietic progenitor cells lose the regulation of tgf--mediated growth inhibition , which in turn increased the incidence of malignant bone tumors .
recent evidence suggests the complexity of the abnormal regulation of p15 gene involved in bone tumor metastasis .
however , the relationship between p15 gene and lung metastasis of osteosarcoma remains to be determined .
this study utilized umr-106 cell line , which is a classical osteosarcoma cell line induced by radioactive phosphorous isotope ( 32-p ) , to generate an osteosarcoma rat model by subcutaneous injection .
the observation of tumor size and pulmonary tissue damage , along with p15 gene expression at different time points after the injection , showed normal tissue structures and almost no tumor cells in the control group . after the inoculation ,
the overall survival rate of rats was 83.3% , in which about 48% of cases had pulmonary metastasis , suggesting the effectiveness and usefulness of this model .
the tumor cell expression maintained at minimal level with lower mutation rate of p15 gene at 1 week after the inoculation . at 26 weeks , however , abnormal tumor - like structures began to occur in the pulmonary tissues in the model group , along with elevated p15 gene mutation rates over time .
the tumor - positive cell percentage in pulmonary tissues increased from 10% to more than 72% at the final time point ( 6 weeks ) , suggesting the occurrence of lung metastasis , as consistent with previous reports .
other studies demonstrated the suppressed incidence of esophagus cancer and pharynx squamous carcinoma by the inhibition of p15 gene methylation .
furthermore , p15 expression level was higher in tongue squamous carcinoma without lymph node metastasis , compared to those patients with metastasis , who also had unfavorable prognosis .
all these studies point to the importance of p15 gene in inhibiting tumor growth and invasion , both of which are consistent with our results in osteosarcoma .
the proliferation of cells is highly dependent on the complete cell cycle , whose disruption or dysfunction may directly affect the tumor growth . p15 gene , as an inhibitory factor in cell - cycle - dependent kinase ( cdk ) , exerts its function mainly on g1 phase of the cell cycle via modulating the n - terminal protein expression of multiple tumor - suppressing factors , thereby inhibiting the abnormal growth of various malignant tumor cells .
the correlation between p15 gene mutation in peripheral blood cells and the growth curve of osteosarcoma lesion suggests the invasion of tumor cells via blood circulation into distal sites , in addition to the facilitating role of mutant p15 in the pulmonary metastasis of osteosarcoma .
our results indicate the close correlation between p15 gene mutation and growth of osteosarcoma , and the potentially direct involvement of p15 gene in pulmonary metastasis .
our results suggest the potency of p15 mutant genes as a novel biological marker in early diagnosis of osteosarcoma in clinics , although its detailed molecular mechanism in progression and pulmonary metastasis require further study . molecular biology research is still required to elucidate the mechanism of mutation location of p15 gene , as well as its role in the metastasis of osteosarcoma to the lung .
 backgroundas a type of primary malignant bone tumor , osteosarcoma has high incidence and poor prognosis , and is predisposed for pulmonary metastasis . 
 the abnormal expression of p15 gene directly participates in the invasion of various cancers . 
 therefore , this study investigated the gene mutation of p15 in both primary lesion and pulmonary metastasis lesion of osteosarcoma in a rat model , in an attempt to elucidate the value of p15 gene as a biological marker.material/methodsa total of 60 sd rats were randomly divided into 2 groups . 
 model rats had injection of osteosarcoma umr-106 cells ( 5106 ) inoculated underneath the right forelimb skin , while control rats received saline injection instead . 
 six rats were sacrificed after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation . 
 tissue samples from inoculation sites and lungs were extracted for measuring the tumor size . 
 sp immunohistochemical ( ihc ) staining was used to detect the positive expression rate , while p15 gene mutation was detected by pcr method.resultswith the elongation of inoculation time , tumor size was significantly increased ( p<0.05 ) . 
 the positive expression rates in both primary and pulmonary metastasis lesions were also significantly elevated ( p<0.05 ) . the occurrence rate of p15 gene mutation in model rats was significantly elevated and showed a correlation with the tumor formation ( r=0.998 , p<0.05).conclusionsthe p15 gene mutation was significantly correlated with osteosarcoma formation and metastasis towards the pulmonary tissue , suggesting its potency as a novel biological marker for early diagnosis of osteosarcoma . 
Background
Material and Methods
Animal model
SP-immunohistochemical (IHC) staining
PCR for P15 gene mutation
Statistical analysis
Results
Tumor size of osteosarcoma lesion
Tumor cell percentage of osteosarcoma and pulmonary metastatic lesions by SP-IHC
P15 gene mutation in rat peripheral blood cells
Discussion
Conclusions