Correlate Study On The Expression Of MiRNA、the Protein Of RASA1 And Human Papillomavirus In Cutaneous Squamous Cell Carcinoma

Objective:In order to study the differential expression of MicroRNA(miRNA) under the condition of human papillomavirus (HPV) infection in cutaneous squamous cell carcinoma (CSCC),we detected HPV in CSCC and investigated the relationship between HPV-positive CSCC and clinical pathology.To test the expression of miR21> 31 in CSCC and to analyze the correlation between HPV infection and miRNA.To observe the expression of p16 and RASA1 protein in CSCC and to explore the carcinogenic mechanism of miR31 in CSCCMethods:Specimens which were 86 paraffin embedded tissues(82 patients) of cutaneous squamous cell carcinoma and 20 cases of normal skin tissue come from the department of pathology in Zhongda Hospital affiliated to Southeast University during 2007.10-2014.10.Among the experimental group 4 cases were two biopsy specimens of the primary tumour and metastatic lymph node.Biopsy specimens were collected from patients (fresh biopsies, n= 10) and adjacent noncancerous tissue samples were used as normal control tissues.Using HPV DNA detection kit and nested PCR to detect HPV in cutaneous squamous cell carcinoma and the control group. We used Quantity One software to determine density value of PCR amplification bands.RT-PCR was used to test the expression of miR21 and miR31 in HPV-positive and HPV-negative CSCC.Finally,we used immunohistochemistry to detect the expression of p16 and RASA1 in CSCC.Results:1 Detect HPV in CSCC1.1 HPV in CSCCIn α-HPV detection by HPV detection kit,there was no difference between cases and control groups of pfraffin embedded tissues.There were one patient in CSCC and none lesion in normal skin.While,In β-HPV detection by nested PCR,15 of 82 (18.29%) patients harboured β-HPV DNA.Only one sample was also positive in control group.Despite the the positive rate of β-HPV in cases was higher than control group’s,there was no significant difference between the two groups.The detection rate of P-HPV DNA was 60%(6/10)in the patients with CSCC in frozen tissues) and 10%(1/10) in the healthy controls(P<0.05), while no significant difference was observed between the two groups.However, HPV detection rate in the frozen tissues was higher than paraffin tissues’.1.2 The association between HPV and clinical pathology in CSCCPatients with HPV infection in CSCC harboured the exposed skin and more than half of them were female patients.Besides that,all of the patients have had a good prognosis so far.But no significant diffierence was found in the infection of HPV among patients of different gender,age,primary sites,size of lesions,duration, lymphatic metastasis groups,and with CSCC of the degree of differentiation, histological grade or clinical stage. 1.3 The association between the viral load of HPV and clinical features in CSCCNo significant diffierence was found in the infection of the viral load of HPV among patients of different gender,age,duration,primary sites,size of lesions,and with CSCC of histological grade or clinical stage (All p>0.05). Viral load was significantly increased in the metastatic CSCC, the CSCC coming from actinic keratosis and SCC in situ.2 The expression of miR21 and miR31 in CSCC2.1 The relative expression level of miR21 and miR31The average relative expression of miR21 in cases was 0.275±0.228,while the expression in normal group was 0.011±0.019.There was a significant difference between the two groups (p<0.001).Similarly, the difference of the average relative expression of miR31 was also statistically significant (p<0.01) between CSCC (0.0129±0.022) and control group (0.0002±0.0002).2.2 The relationship between the expression level of miR21、miR31 and clinical pathology in CSCCNo significant differences were observed between high and low miR-21 expression and age,gender,duration,the primary tumor site,tumor size,histological grade,clinical stage,lymph node metastasis,depth of invasion,and HPV infection in CSCC.However,the expression level of miR21 was positively correlated with the degree of differentiation (p<0.05,r2=0.19).The expression of miR31 in CSCC had no significant correlation with clinical and pathological characteristics.2.3 Correlation between HPV infection and miR21> miR31 in CSCCThe expression of miR21 in HPV-positive CSCC was higher than HPV-negative group;but the difference was not statistically significant. However,the expression level of miR31 was significantly decreased in HPV-positive CSCC (p<0.01).The expression of miR31 in HPV-positive group correlated with age (p= 0.01).The patients equal or over 60 years old in HPV-positive CSCC have higher expression of miR31 than younger patients.3 The expression of p16、RASA1 protein in CSCC3.1 The expression of p16P16 expression was found in 59 out of 82 CSCC;while,all of the p16 expression in control goup were positive (p=0.017).The expression level of p16 was significantly related to the differentiation of CSCC (p=0.001),pathological grade (p=0.001),and clinical stage (p=0.044).3.2 The expression of RASAlA total of 15 HPV-positive cases,17 HPV-negative CSCC and 10 normal samples were chosen to test RASA1.There were 20 cases of negative,7 cases of weak positive,4 samples of positive and 1 case of strongly positive in CSCC group;while,no negative expression was found in control group.There was a significant difference in the two groups (p<0.001).P16 expression was negative correlated with the expression level of miR31 in HPV-negative tissues (p<0.05, r=-0.588).Conclusion:1 The positive rate of HPV in CSCC is not high;the most frepuent species in it is P-HPV.There is a high viral load in the metastatic CSCC,the CSCC coming from actinic keratosis and SCC in site.HPV may play a role in the early stage of tumor.2 The expression level of miR21 and miR31 is high in CSCC.The expression of miR21 was positive correlated with the degree of differentiation,and there is a correlation between the expression of miR31 and HPV infection.We consider miR21、31 are both tumor-associated MicroRNAs,and miR.31 may be a kind of HPV-associated MicroRNA.3 There is a negative correlation between miR-31 expression and RASA1 protein expression in HPV-negative CSCC. The high expression of miR31 may promote the proliferation of cells through down-regulation of RASA1 protein expression.