| Soft tissue sarcomas(STS)are rare malignant tumors of mesenchymal origin with high aggressiveness and heterogeneity.More than 50 histologic subtypes of STS have been identified.Incidence of soft tissue sarcomas is account for 0.7%～0.8%in adult malignant tumors,and 5%～7%in children malignant tumors.Pathological subtypes of soft tissue sarcomas are complicated and diverse,and the incidence and common pathological types of soft tissue sarcomas in different geographical areas are different.But there is no report on incidence characteristics of soft tissue sarcomas in Henan province in China.The incidence rate of soft tissue sarcomas is low,but they are malignant,grow quickly and infiltratedly,easy to metastasis and reoccurance.However,its etiology and mechanism are still unknown.It is meaningful to explore the mechanism of the initiation of soft tissue sarcoma,which could provide effective evidence for prevention and treatment.Epidemiological investigation showed that genetic susceptibility is associated with the occurrence of soft tissue sarcoma.Antioncogenes mutations were found in sarcoma tissues from patients with soft tissue sarcoma,for example Neurofibromin 1(NF1),p16Ink4a and PTEN(Phosphatase and tensin homolog deleted on chromosome ten).If antioncogenes mutate,the expression of tumor suppressor protein encoded from antioncogenes may change to induce tumorgenesis,which could not inhibit cell proliferation or regulate signaling pathways of cell cycle.But there are only few reports on the protein expression of p16Ink4a PTEN in soft tissue sarcoma,and even no report on the analysis of NF1 protein expression alone,or the expression of three tumor suppressor protein together in soft tissue sarcoma.MicroRNAs(miRNAs)are a class of endogenous non-coding RNAs that could regulate genes.The change of miRNAs expression is associated with the occurrence and development of many kinds of tumors.miRNAs not only act on oncogenes,but also act on antioncogenes.Studies demonstrated that there were some miRNAs could regulate NF1,p16Ink4a,PTEN,but it is still unclear that which miRNAs regulate NF1,p16Ink4a or PTEN in the initiation of soft tissue sarcoma.Objective1.To analyze the characteristics of 1624 soft tissue sarcoma cases during 2006 to 2016 in Henan Province Cancer Hospital,and give a clue on the prevention,treatment and research in the field of soft tissue sarcoma in Henan.2.Undifferentiated pleomorphic sarcoma(UPS)with the highest constituent ratio in soft tissue sarcoma is as the key on research.In order to explore the protein expression of antioncogenes NF1,p16Ink4a,PTEN and the important protein of the involved signal pathways in UPS,find out the miRNAs regulating the three antioncogenes and with differential expression in UPS,which could provide evidence and clue for the occurrence of soft tissue sarcoma.3.To set up NF1/p16Ink4a/PTEN spatially and temporally restricted mouse model of soft tissue sarcoma,verify the role of NF1,p16Ink4a and PTEN in UPS,verify and analyze related miRNAs expression in mice UPS,which could provide a good platform for studying the initiation,development and metastasis of soft tissue sarcoma.Methods1.The information of electronic medical record from the patient with soft tissue sarcoma in Henan Province Cancer Hospital(Cancer Hospital Affiliated of Zhengzhou University)for the first time during January 1,2006 to December 31,2016 was collected,including hospitalization number,gender,age,date of birth,place of birth,admission date,discharge date,tumor location,pathological type et al.Descriptive statistics was analyzed on age,gender,pathological type,days of hospitalization,place of birth and tumor location,and constituent ratio of different pathological subtypes was calculated using SPSS21.0 software.2.UPS with the highest constituent ratio in soft tissue sarcoma is as the key on research.68 pairs of UPS sarcoma tissues and adjacent normal muscle tissues were collected from surgical resection in Department of bone and soft tissue sarcoma in Henan Province Cancer Hospital.The protein expression of NF1,p16Ink4a,PTEN was detected using immunohistochemistry,and the ratio of two or three low-expressed protein was analyzed.Then the important phosphorylated protein expression of p-Akt,p-mTOR,p-S6 and p-Erk in the three antioncogenes-involved signal pathways in UPS sarcoma tissues and adjacent tissues was determined using immunohistochemistry,the combination of CDK6 and Cyclin D1 was detected using immunofluorescence double staining and confocol.Furthermore,the differential expression of miRNAs between UPS sarcoma tissues and adjacent tissues was screened and analyzed using miRNA sequencing,and the miRNAs which were expressed differentially and regulated NF1,p16Ink4a,PTEN were screened and verified by qPCR.3.Base on the theory of Cre/LoxP,NF1flox/flox-Ink4a/Arflaox/flox-PTENflox/flox mice were used to set up a spatially and temporally restricted mouse model of soft tissue sarcoma.An adenovirus expressing Cre recombinase(Ad-Cre)was injected in the muscle of left hindlimb of mice,and the soft tissue sarcoma were generated.Latent period of sarcoma formation,sarcoma number,and metastasis was observed,and the rate of sarcoma was calculated.The protein expression of NF1,p16Ink4a,PTEN,p-Akt,p-mTOR,p-S6,p-Erk in mice sarcoma tissues and adjacent tissues was detected using imunohistochemistry.The combination of CDK6 and Cyclin D1 was detected using immunofluorescence double staining and confocol.The miRNAs regulating NF1,p16Ink4a,PTEN were verified in mice model using qPCR.Results1.(1)There were 1624 patients with soft tissue sarcoma in Henan Province Cancer Hospital in 2006～2016,the average age of patients was 44.71±17.91.The number of soft tissue sarcoma patients increased significantly since the age of 15,reached the peak at the age of 55 to 65,and finally declined rapidly after the age of 75.(2)The top nine pathological subtypes of soft tissue sarcoma with high constituent ratio were undifferentiated pleomorphic sarcoma(23.83%),synovial sarcoma(16.69%),liposarcoma(13.67%),fibrosarcoma(10.22%),sarcoma without definite type(8.99%),leiomyosarcoma(7.02%),dermatfibrosarcoma protuberant(5.79%),rhabdomyosarcoma(4.68%)and malignant peripheral nerve sheath tumor(4.25%),accounting for 95.15%of total number of inpatients of soft tissue sarcoma.(3)In total 1624 inpatients of soft tissue sarcoma,the male and female inpatients were 923 and 701 respectively.The gender ratio was 1.32:1.The top three pathological subtypes of soft tissue sarcoma in either male or female inpatients with high constituent ratio were undifferentiated pleomorphic sarcoma(UPS),synovial sarcoma,liposarcoma.(4)The change of the top nine pathological subtypes of soft tissue sarcoma in Henan Province Cancer Hospital showed that,the rank of UPS kept the first from 2006 to 2016;The rank of liposarcoma was increased(4th to 2nd,from 2006 to 2016),and the rank of fibrosarcoma was decreased(3rd to 5th,from 2006 to 2016).(5)The most inpatients with soft tissue sarcoma came from eastern area of Henan province,accounting for 28.57%of total number of inpatients,the second most inpatients from central area of Henan(24.57%),and the constituent ratio of inpatients from southern,northern and western Henan were 21.12%,13.49%and 10.34%,respectively.2.(1)In 68 cases of UPS sarcoma tissues,the positive rates of NF1,p16Ink4a and PTEN protein were 39.71%,55.88%and 10.29%.And in adjacent tissues,the positive rates of NF1,p16Ink4a and PTEN protein were 70.59%,75.0%and 94.12%,respectively.There were significant statistically differences on the positive rates of NF1,p16Ink4a and PTEN protein in UPS sarcoma tissues compared to those in adjacent tissues(P<0.05).In addition,there was no significant difference on the protein expression of NF1,p16Ink4a or PTEN in UPS sarcoma tissues between male and female,or between the age of<55 and the age of ≥55(P>0.05).Furthermore,in 68 UPS patients,4 patients(5.88%)were found with NF1(-)pl6Ink4a(-)PTEN(+),4 patients(5.88%)with NF1(+)p16Ink4a(-)PTEN(-)in sarcoma tissues,respectively;14 patients(20.59%)were found with NF1(-)p16Ink4a(+)PTEN(-)in sarcoma tissues;And 19 patients(27.94%)were found with low or no expression of NF1(-)p16Ink4a(-)PTEN(-).(2)The ratios of positive area to total area of p-Akt,p-mTOR,p-S6 and p-Erk in UPS sarcoma tissues were(17.444±2.73)%,(18.32±1.90)%,(22.52±2.39)%and(14.25±1.55)%,which were significantly increased compared to those in adjacent tissues((6.41±1.60)%,(7.25±1.74)%,(4.29±1.50)%,(5.69±1.88)%,respectively)(P<0.05).And the rate of positive cell of the combination of CDK6 and Cyclin D1 in UPS sarcoma tissues was(19.50±3.66)%,but there was no positive cell in adjacent tissues.(3)The miRNAs with differential expression between UPS sarcoma tissues and adjacent tissues were screened and analyzed using miRNA sequencing.Base on more restrictive criteria:more than 50 copies in UPS sarcoma and adjacent tissues,a fivefold difference in expression in UPS sarcoma tissues relative to the expression in adjacent tissues,and P value of less than 0.05,125 miRNAs were screened,of which 82 were upregulated and 43 downregulated in UPS sarcoma tissues.Target genes were predicted using Targetscan and miRanda software,and the miRNAs were screened reversely which may regulate NF1,p16Ink4a or PTEN and were differentially expressed.Eight miRNAs were chosen and verified in 68 UPS sarcoma and adjacent tissues:miR-127-3p,miR-199a-3p and miR-34a-5p(regulating NF1)5 miR-503-5p and miR-497-5p(regulating p16Ink4a),miR-21-3p、miR-378d and miR-378a-3p(regulating PTEN).There was no significant difference on the relative expression of miR-127-3p in UPS sarcoma tissues compared with adjacent tissues(P>0.05).The relative expressions of miR-199a-3p,miR-503-5p,miR-21-3p in UPS sarcoma tissues were 4.10±0.77,6.60±1.61,4.34±0.79,which were significantly upregulated compared to those in adjacent tissues(1.02±0.22,1.24±0.36,1.23±0.30,respectively)(P<0.05);The relative expressions of miR-34a-5p,miR-497-5p,miR-378d,miR-378a-3p in UPS sarcoma tissues were 0.19±0.95,0.21±0.04,0.0041±0.0012,0.0054±0.0013 respectively,which were significantly downregulated compared to those in adjacent tissues(1.31±0.38,1.177±0.30,1.74±0.65,1.91±0.86,respectively)(P<0.05).3.(1)All of 45 NF1flox/flox-Ink4a/Arfflox/flox-PTEN flox/flox mice generated soft tissue sarcoma at the Ad-Cre injection site,sarcoma rate was 100%.Latent period of sarcoma formation was(53.4±3.1)day,3 of 45 mice showed metastatic lung cancer.All the sarcomas were pathologically identified as undifferentiated pleomorphic sarcomas(UPS).(2)In mice model,there was no protein expression of NF1,p16Ink4a or PTEN in UPS sarcoma tissues,but the NF1,p16Ink4a and PTEN protein were all positive in adjacent tissues.The ratios of positive area to total area of p-Akt,p-mTOR,p-S6 and p-Erk in mice UPS sarcoma tissues were(41.34±1.61)%,(24.27±3.89)%,(10,87±1.34)%,(10.56±2.50)%,which were significantly increased compared to those in adjacent tissues of mice((8.31±1.91)%,(7.64±1.88)%,(2.24±1.14)%and(2.61±1.21)%,respectively)(P<0.05).And the rate of positive cell of the combination of CDK6 and Cyclin D1 in mice UPS sarcoma tissues was(8.30±1.08)%,but there was no positive cell in adjacent tissues in mice.(3)Eight miRNAs screened from human study were verified in 45 pairs of UPS sarcoma and adjacent tissues in mice model.There was increased trend of miR-127-3p,miR-199a-3p and miR-503-5p in mice UPS sarcoma tissues,but there was no significant difference compared with those in mice adjacent tissues(P>0.05);The relative expressions of miR-34a-5p,miR-497-5p,miR-21-3p,miR-378d and miR-378a-3p in mice UPS sarcoma tissues were 0.03±0.01,0.12±0.03,0.04±0.02,O.0093±0.0052 and 0.0040±0.0021 respectively,which were significantly downregulated compared to those in mice adjacent tissues(1.55±0.60,1.13±0.38,1.47±0.47,1.22±0.31 and 1.49±0.50,respectively)(P<0.05).Conclusions1.There are 1624 patients with soft tissue sarcoma in Henan Province Cancer Hospital in 2006～2016,the average age of patients is(44.71±17.91)year.The gender ratio(male:female)is 1.32:1.The top three pathological subtypes of soft tissue sarcoma with high constituent ratio are undifferentiated pleomorphic sarcoma(UPS),synovial sarcoma,liposarcoma.And the rank of UPS keeps the first from 2006 to 2016.UPS should be paid more attention on the prevention,treatment and research in Henan in future.2.Low/no protein expression of NF1,p16Ink4a and PTEN alone/together,and the activation of the involved Ras-Raf-Mek-Erk,PI3K-Akt-mTOR-S6,CDK6-Cyclin D1 signal pathway are associated with the occurrence of UPS.The miRNAs,which are expressed differentially and regulate antioncogenes of NF1,p16Ink4a and PTEN,are screened using miRNA sequencing and bioinformatics:miR-199a-3p,miR-503-5p and miR-21-3p are upregulated significantly,miR-497-5p,miR-378d and miR-378a-3p are downregulated significantly in sarcoma tissues.3.A new and optimized spatially and temporally restricted mouse model of soft tissue sarcoma is set up successfully.In this mouse model,the protein expression of NF1,p16Ink4a and PTEN and the important phosphorylated protein expression of the three antioncogenes-involved signal pathways are veirified.The miRNAs are no changed miR-127-3p,miR-199a-3p and miR-503-5p,and downregulated miR-21-3p,miR-34a-5p,miR-497-5,miR-378d and miR-378a-3p in mice sarcoma tissues. |
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