Study On The Effect Of Serum Matrix Metalloproteinase -2/-9 And Its Gene Polymorphism On The Pathogenesis,Development And Prognosis Of Cervical Cancer

Backgroud and ObjectiveCervical cancer(CC)is the second common malignant tumor in women,and is also the third major cause of cancer death in women in developing countries.Cervical intraepithelial neoplasia(CIN)is a precancerous lesion of cervical cancer.It is a general term for a series of cervical squamous epithelial lesions,and it can develop into squamous invasive carcinoma.The occurrence and development of cervical cancer is a rather complex biological process,which is related to many factors.Human papillomavirus(HPV)infection is the main risk factor of cervical cancer.The invasion of cervical cancer needs to break.through the basement membrane and be closely related to the extracellular matrix(ECM).In ECM,matrix metalloproteinases(MMPs)is a member of metalloendopeptidases.It can hydrolyze various proteins in ECM,destroy cell barrier and basement membrane,and promote the migration and invasion of tumor cells.MMPs,especially MMP-2 and MMP-9.are overexpressed in a variety of tumor tissues,and play an important role in the formation and development of cancer.In recent years,studies have shown that MMP-2 and MMP-9 are overexpressed in cervical cancer and precancerous lesions,and are of potential value in the diagnosis and prognosis of cervical cancer.Some evidences show that the expression and activity of MMP-2 and MMP-9 are closely related to the development of the normal cervix to CIN and eventually to the process of cervical cancer.However,previous studies on the relationship between MMP-2 and MMP-9 and cervical cancer are one-sided.Most of them use the method of tissue immunity to detect the expression of MMPs,which is cumbersome in operation.In addition,the clinical study of the relationship between MMP-2 and MMP-9 with cervical cancer in Chinese population,and the specificity and sensitivity of the diagnosis and prognosis are rarely reported.Therefore,the first part of this study aims to analyze the MMP-2 and MMP-9 in normal and CIN and cervical cancer in serum expression,to explore the possibility for detection of cervical cancer biomarkers of serum ELISA.Meanwhile,we explored the relationship between the expression level of MMP-2 and MMP-9 and the pathogenesis,progression and prognosis of CIN and cervical cancer,and explored the possibility of it as a diagnostic,developing and prognostic index and risk factor for cervical cancer.Gene polymorphism plays an important role in a variety of pathophysiological processes,including cancer.Among them,single nucleotide polymorphisms(SNPs)is the most common genetic variation of human genes,and is considered to be the new generation of genetic markers.It has been shown that the SNP polymorphism of multiple genes is closely related to the genetic susceptibility due to the regulation of DNA mismatch repair,cell cycle,metabolism,and immunity.The gene promoter SNP can regulate the transcriptional activity of the gene by acting on various transcription factors.Studies have shown that MMP-2 promoter region SNP rs2285053,rs243865 and MMP-9 rs3918242 polymorphism can affect the transcriptional activity of genes and alter the level of protein expression,which is closely related to the genetic susceptibility of tumors.However,there are few researchs on the relationship between the MMP-2 and MMP-9 promoter SNPs polymorphism and the risk of cervical cancer.Therefore,the second part of this study is to explore the genotype distribution of MMP-2 rs2285053 and MMP-9 rs3918242 polymorphism,and study the relationship between the genetic susceptibility,development and prognosis of cervical cancer,and to analyze the interaction of HPV infection,MMP-2 rs2285053 and MMP-9 rs391 8242 polymorphism.Methods1.The relationship of MMP-2 and MMP-9 with cervical cancer and its precancerous lesions.A total of 86 female outpatients and hospitalized patients in June 2013 to September2014 were selected,which including 35 cases of cervical cancer,51 cases of CIN.And 29 cases of normal cervical epithelium were selected as control group.The general data of cases and control were collected,venous blood was collected,and the expression levels of MMP-2 and MMP-9 in three groups of-serum samples were analyzed by ELISA.The risk factors of CIN and cervical cancer(age and HPV infection)were analyzed.The serum MMP-2 and MMP-9 levels of the three groups were compared.The receiver operating characteristic curve(ROC)analysis used for the sensitivity and specificity of occurrence,classification and pathogenesis of CIN,as well as clinical staging,metastasis,recurrence and prognosis prediction of cervical cancer.Multiple regression analysis of the correlation between the two with CIN and cervical cancer.2.MMP-2 and MMP-9 gene polymorphisms associatedwith cervical cancer risk.The cases were 150 CC patients aged 22-75and the controls were 120 healthy objects aged20-79.The cases wereselected fromSeptember,2010 to December,2014 and werepathologically diagnosed with cervical cancer.The controls were randomlyrecruited from healthy individuals who got regularcheck-ups in the corresponding hospital.Allthe subjects agreed to provide 5 ml freshperipheral blood.Restriction fragment length polymorphism-polymerase chain reaction(PCR-RFLP)was used to analyze the genotypes and alleles of the samples.Statistical analysis and comparison of the frequencies and allele frequencies of the polymorphic genotype of MMP-2 rs2285053 and MMP-9 rs3918242 in two groups of cases.The correlation of cervical cancer and its staging and lymph node metastasis with these two SNPs was analyzed by multiple regression analysis.The effects of the interaction of HPV infection,MMP-2 and MMP-9 SNPs polymorphisms on the susceptibility,metastasis andrelapse of cervical cancer were analyzed by the regulation effect regression model.Results1.The incidence of HPV infection in patients with cervical cancer(77.1%)was significantly higher than that of patients with CIN(54.9%)and normal control group(27.6%)(P<0.05).HPV infection is a risk factor for CIN and cervical cancer(CIN:OR=3.196,P=0.021,95%CI=1.195-8.545,cervical cancer:OR=5.123,P=0.010,95%CI=1.482-17.718).The level of serum MMP-2 and MMP-9 in the patients with cervical cancer was significantly higher than that in group CIN(P=0.000)and normal group(P=0.000).The diagnostic specificity and sensitivity of the MMP-2 and MMP-9 for the patients with CIN and cervical cancer were statistically significant(AUC>0.648,P<0.05),and overexpression of the two proteins is a risk factor for CIN and cervical cancer(P<0.008,OR>3.28).Subgroup analysis showed that the higher the CIN levels wasaccompanied by higher MMP-9 and MMP-2 levels.The level of MMP-2 and MMP-9 in serum could be used as a diagnostic index of CIN grading(P<0.05).And with the increase of serum concentration of MMP-2 and MMP-9,the risk factor of HSIL in patients increased(OR>1.98,P<0.05).With the increase of the stage of cervical cancer,the serum concentration of MMP-2 and MMP-9 increased(P<0.05).Although the two proteins were not highly effective in the diagnosis of stageⅠ and Ⅱ of cervical cancer(AUC<0.55,P>0.05),it could be used as a diagnostic index for the stage of cervical cancer(P<0.05).the risk factor of tumor invasion and metastasis in cervical cancer patients increased(OR>3.18,P<0.05)accompanied by the increase of MMP-2 and MMP-9 levels(OR>3.18,P<0.05),The development of cervical cancer patients with CIN had a significantly higher level of MMP-9 expression than those who did not develop cervical cancer,but MMP-2 level had no significant difference(P>0.05).When the serum MMP-9 of CIN patients is greater than 172.26 ng/mL,the possibility of developing cervical cancer within 3 years is more likely(P=0.009).When the concentration of MMP-9 is greater than 147.1 ng/mL,the risk of the evolution of CIN(mainly CIN II/III)to cervical cancer is significantly increased(OR=2.73).The serum initial MMP-2 and MMP-9 levels were significantly higher in patients with recurrent cervical cancer than in patients with non recurrent cervical cancer(P=0.002 and 0.024,respectively).When MMP-2 was greater than 74.16 ng/mL or MMP-9 was greater than 203.22 ng/mL,the recurrence rate of cervical cancer was greater in 3 vears(MMP-2:AUC=0.801.P=0.003,MMP-9:AUC=0.720,P=0.031).The higher the initial serum MMP-2 and MMP-9 levels,the higher the risk of recurrence of cervical cancer.(MMP-2:P=0.001,corrected OR=4.58,95%CI=1.02-19.25;MMP-9:P=0.01 7,corrected OR=2.83,95%CI=0.77-14.63).In addition,patients with higher MMP-2 and MMP-9 levels had higher risk of CIN and cervical cancer.HSIL,CIN to cervical cancer,cervical cancer invasion,metastasis and recurrence(OR>2).The median survival time of patients with serum initial MMP-2 and MMP-9 low expression was significantly higher than those with high expression(P=0.000 and 0.000,respectively).2.The genotype frequencies(CC,CT and TT)of MMP-2 rs228505 in 150 cases of cervical cancer were 77.3%.22.7%and 0%,respectively,and allele frequencies(C and T)were 88.7%and 11.3%,respectively.The genotype frequencies of MMP-9 rs3918242(CC,CT and TT)were 77.3%,20%and 2.7%respectively,the allele frequency(C and T)was 86%and 14%,respectively.The differences of genotype distribution frequencies of the two SNPs between the two groups were not apparent(P>0.05),and no significant relevance was found betweenthe two SNPs and the lymphatic metastasis,clinical stage,relapse and life span of cervical cancer.Interaction analysis results show that the interaction between MMP-2 rs2285053 and MMP-9 rs3918242 had a significant relationship with cervical cancer susceptibility(OR=0.135,95%CI=0.028-0.660),lymph node metastasis(OR=0.094,95%=0.013-0.467)and tumor recurrence(OR=0.115,95%=0.019-0.738).The interaction between MMP-2 SNP polymorphism and HPV infection was significantly related to the susceptibility of cervical cancer(OR=0.147,95%CI=0.038-0.594),but was not related to lymph node metastasis and recurrence of cervical cancer(P>0.05).The interaction between MMP-9 SNP polymorphism and HPV infection was significantly related to cervical cancer susceptibility(OR=2.044,95%CI=1.124-4.698),lymph node metastasis(OR=3.359,95%CI=1.171-5.643)and recurrence(OR=0.148,95%CI=0.363-2.495).Conclusion1.HPV infection is a major risk factor for the occurrence of CIN and cervical cancer.MMP-2 and MMP-9 are significantly correlated with CIN and the susceptibility and risk of cervical cancer occurrence,development and prognosis,and can be used as serological biomarkers.The diagnostic efficiency of MMP-9 is higher than that of MMP-2.2.The polymorphism of MMP-2 rs2285053 and MMP-9 rs3918242 gene are not correlated with the risk of the susceptivity,development and prognosis of cervical cancer,but the interaction among the three HPV infection,MMP-2 and MMP-9 gene polymorphisms is related to the occurrence,development and prognosis of cervical cancer.