| Cervical cancer is one of the most common malignant tumor in gynecology, and it has become a serious public health problem that being harmful to women’s physical and mental health. It is well known that persistent high risk HPV infection is the major etiology of cervical cancer, but not the only factor. Folate receptor alpha(FRα) is high expressed in cervical cancer cell, and associated with cervical carcinogenesis, however, the relationship and underling mechanisms are not clear precisely. The study will evaluate the effects and possible molecular biology mechanisms of FRα in cervical carcinomas development and progression in vitro and in vivo, and may provide evidences for etiology and pathogenesis and prevention and treatment of cervical cancer. Objective:The aim was to evaluate the effects and possible molecular biology mechanisms of FRα through mediating ERK signaling pathway in progression of cervical carcinomas in vitro and in vivo, and the results might provide evidences for etiology and pathogenesis of cervical cancer and afford novel insights into the cancer management. Methods:Epidemiological study: 176 patients were involved in the present study, including 34 cervix inflammation and 26 low degree cervical intraepithelial neoplasia(CINⅠ) and 57 middle and high degree cervical intraepithelial neoplasia(CINⅡ/CINⅢ) and 59 cervical cancer. All subjects were investigated with the same structured questionnaire, which included demographic characteristics and personal hygiene habits and reproduction status. Their cervical tissues were collected from operation and biopsy. HPVs expression status were tested with PCR. FRα and p-Src and p-ERK1/2 and p-c-Fos and p-c-Jun protein levels were evaluated with Immunohistochemical method.Cell study in vivo: Firstly, FRα protein expression levels were measured by flow cytometry(FCM) for the cervical cancer cells of Hela and Caski and Siha and C33 A. Then FRα protein level was down-regulted with si RNA interference for the FRα strongest positive Hela cells. And p-Src and p-ERK1/2 protein levels were down-regulted respectively with Src inhibitor PP2 and ERK1/2 inhibitor U0126 for Hela and C33 A cells. Cell viability was determined by CCK-8 assay; cell cycle and apoptosis were detected by flow cytometry; m RNA expression levels of FRα and Src and ERK1 and ERK2 and c-Fos and c-Jun were tested by Real-time PCR; and their total and phosphorylated protein levels were measured by Western-blot.The database was established and analysed by SPSS17.0 statistical software and with t test, χ2 test, χ2 trend test, ANOVA, Repeated Measurement of General Linear Model and LSD-t test for comparison two groups for signal factor analysis, and the multivariate analysis was performed with Multinomial Logistic Regression. Results:1. χ2 test in epidemiological study showed that age between 45 to 55(χ2=25.308,P=0.003), junior cultural level(χ2=11.854,P=0.008), live in rural(χ2=19.141,P<0.001), HPVs infection(χ2=19.44,P=0.001), low frequency for bath(χ2=17.067,P=0.009), low frequence for clear vagina(χ2=17.238,P=0.008), the first time menarche after 18 years old(χ2=23.775,P=0.001), age at first sexual intercourse after 25 or before 16 years old(χ2=16.356,P=0.012), gynecological disease(χ2=13.988, P=0.003), production time more than 4 times(χ2=15.187,P=0.019) were risk factors for cervical carcinomas.2.Following the cervical lesions degree increase,the strongest positive expression(++~+++)rates of FRα(χ2=8.848,P=0.031;χ2趋势=4.965,P=0.026)and p-c-Jun(χ2=15.719,P=0.001;χ2趋势=13.253,P<0.001)and the positive expression(+~+++)rates of p-Src(χ2=12.62,P=0.006;χ2趋势=9.49,P=0.002)and p-ERK1/2(χ2=11.00,P=0.012;χ2趋势=10.58,P=0.001)and p-c-Fos(χ2=13.62,P=0.003;χ2趋势=10.39,P=0.001)protein increased.3. After down-regulation FRα or p-Src or p-ERK1/2 protein level, cell proliferation was inhibited, and apoptosis was promoted, and the cell cycle was arrested in G0/G1 stage while the proportion of S and G2/M stages and PI were decreased(P<0.05). The changes of cell proliferation and cell cycle and PI between Hela and C33 A cells after Src inhibition had no statistical difference(P>0.05), and the changes had no statistical difference after ERK1/2 inhibition too(P>0.05). The changes of apoptosis rate decrease of Hela were lower than C33 A after inhibit Src(t=-31.631,P<0.001) and inhibit ERK(t=-0.724,P=0.496).4. After down-regulation FRα of Hela cell, the m RNA levels of Src and ERK1 and ERK2 and c-Jun increased(P<0.05), the protein levels of p-ERK1/2 and c-Fos and p-c-Fos and c-Jun and p-c-Jun decreased(P<0.05), while p-Src protein level increased(P<0.05). After down-regulation Src of Hela and C33 A cells,the m RNA levels of ERK1 and ERK2 and c-Fos and c-Jun increased(P<0.05), the protein levels of ERK1/2 and p-ERK1/2 and p-c-Fos decreased(P<0.05), while c-Jun and p-c-Jun protein levels increased(P<0.05). After the inhibition of ERK in Hela and C33 A cells,the m RNA levels of c-Fos and c-Jun of Hela cell increased(P<0.05), the protein level of p-c-Fos decreased and p-c-Jun increased both in Hela and C33A(P<0.05).5. Multivariate analysis showed that bathing frequency > 1 times/week(OR=0.026) was protect factor for CINⅠ,gynecological disease(OR=8.904) and FRα(OR=6.599) were risk factors for CINⅠ; bathing frequency > 1 times/week(OR=0.088) was protect factor for CINⅡ/Ⅲ, and HPVs infection(OR=5.588) was risk factor for CINⅡ/Ⅲ;live in rural(OR=6.387) and HPVs infection(OR=12.894) and FRα(OR=8.210) and p-Src(OR=5.236) and p-c-Jun(OR=4.281) were risk factors for SCC. Conclusions:1. FRα and Src and ERK1/2 and c-Fos and c-Jun proteins could promote the development and progression of cervical cancer. FRα and Src and ERK1/2 could promote cell proliferation and inhibit apoptosis and promote cell cycle to G2/M stage of cervical cancer cells.2. FRα might promote cervical carcinogenesis progression through inactiving key factors of ERK1/2 and c-Fos and c-Jun in ERK signaling pathway. Src might play roles in cervical carcinogenesis through up-regulation ERK1/2 and c-Fos protein levels. ERK might affect cervical cancer progression through inactiving c-Fos protein expression. FRα might promote cervical carcinogenesis through activating the key factors of ERK signaling pathway. It was implied that FRα could be a new target for prevention and treatment of cervical cancer.3. After down-regulation FRα, p-Src protein level increase; after inhibit Src and ERK, p-c-Jun protein level increase, it might be correlated with other factors in signaling pathway or might be correlated with negative feedback, but the mechanisms should be researched further.4. HPVs infection and high level of FRα and live in rural and p-Src and p-c-Jun protein and gynecological disease were the major risk factors for cervical cancer and CIN,. Good hygienic habit such as bath was protect factor for cervical cancer lesion. |
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