The Effect And Mechanism Of Folate And MiR-224 Regulating Autophagy In Cervical Cancer Cells

Cervical cancer is one of the most common malignant tumors of the reproductive tract in the world.There are about 500 thousand new cases in the world,of which more than 90% occured in developing countries.China’s new cases each year accounted for nearly 1/3 of the world’s total cases.The study found that cervical intraepithelial neoplasia(CIN)is a precervical cancer lesions,which can progress to cancer,can also naturally subsided.High-risk human papillomavirus(HR-HPV)persistent infection is an important factor in the development of cervical cancer and CIN progress for cervical cancer,but only about 1% of HR-HPV infected women eventually develop to cervical cancer,and it often takes about 8-10 years from CIN to cervical cancer.High risk human papillomavirus infection is one of the main causes of cervical cancer,but it is not the only risk factor.There may be other factors played a synergistic effect in the occurrence and development of cervical cancer.Therefore,to explore these synergistic factors have important significance in elucidating the occurrence and development of cervical cancer and cervical cancer prevention.A study found that folic acid supplementation in women with HR-HPV(especially HPV-16)may reduce the risk of cervical intraepithelial neoplasia(CIN)and worse.miRNA can regulate cell proliferation,apoptosis,differentiation,metabolism,carcinogenesis and autophagy.As a member of micro-RNAs family,miR-224 plays an important role in the occurrence and development of tumor cells,which is involved in the process of tumor cell proliferation,differentiation,apoptosis,invasion and metastasis.miR-224 is highly expressed in some tumors,such as liver cancer,breast cancer,colorectal cancer,cervical cancer and so on.miR-224 promotes or inhibits tumor cell invasion or metastasis by acting on different target genes in different cancers.It has been found that miR-224 can be inhibited by Smad4 B virus related hepatocellular carcinoma cell autophagy,there is evidence that chronic inhibition of autophagyin normal tissues,can activate tumor formation process,the local inflammatory response inhibition of autophagy can aggravate the necrotic cells,which lead to the growth of tumor.At the same time,autophagy also plays a role against tumor cells,autophagy may be closely related with the occurrence and development of tumor cells.Autophagy related protein Beclin1 is a key protein promoter of autophagy,was the first to be confirmed,and autophagy activation is directly related to the tumor suppressor gene Beclin1 deletion caused by tumor.LC3 is the only protein that has been found to be involved in the regulation of signal transduction,and is a good marker of autophagy.It is reported that autophagyin mouse embryonic stem cells can be induced by folate deficiency,and the expression of autophagy related genes is up-regulated,which promotes the occurrence of autophagy.However,the effect and mechanism of miR-224 on autophagy in cervical cancer cells have not been reported.This project aims to study whether autophagy of cervical cancer cells is affected by folate deficiency and it’s mechanism.It is helpful to provide theoretical basis for elucidation of the pathogenesis and prevention of cervical cancer.PartⅠ Risk factors for cervical intraepithelial neoplasia in Chinese women: large study in Jiexiu,Shanxi province,ChinaObjective: To explore the risk factors of cervical intraepithelial neoplasia(CIN)in women: in Jiexiu,Shanxi,China,and to provide the basis for the prevention of cervical cancer.Twenty thousand women were selected from 82 villages,towns,and mining regions in the Jiexiu area,Shanxi province,China,between June 2014 and November 2014.All the women volunteered to participate in the study.The selection criteria were as follows:married women aged 18–65 years,women of the Han ethnicity,and women who had resided in Jiexiu for at least 1 year.All the subjects had volunteered to participate in the study and signed informed consent.Study visit 1:Enrollment visit for all participants.The 20,000 eligible women were administered a questionnaire onthe potential risk factors for CIN and underwent the pap test usingliquid based cytologyanalysis.Study visit 2 : All women with abnormal pap test results(ASC-USor worse)were referredfor colposcopy with biopsy and/or endocervicalcurettage(ECC)according tothe same protocol.The women with negative or inadequate colposcopic findings alsounderwent ECC.The women were divided into the study group and the control group based on the histological findings.The study group consisted of the women with histological CIN1,CIN2/3,whereas the control group consisted of the women with negative histological results who volunteered to participate in the follow-up study.Dietary survey,vaginal microflora detection and human papillomavirus(HPV)were conducted for these women.Logistic regression analysis was used to analyze the risk factors of CIN.Methods:Results:(1)Histology and pathology: 1438 cases of cervical cytology in 20000 cases were detected in ASC-US and worse(including abnormal cervical glandular cells),accounting for 7.19%,including 1,270 women(6.35%)with ASC-US,7 women(0.04%)with atypical squamous cells,cannot exclude high-grade squamous intraepithelial lesion(ASC-H),125 women(0.63%)withlow-grade squamous intraepithelial lesion(LSIL),32women(0.16%)with high-grade squamous intraepithelial lesion(HSIL),2 women(0.01%)with squamous cervical carcinoma(SCC),and 2 women(0.01%)with atypical glandular cells(AGC).A total of 18,562 women(92.81%)women tested negative for intraepithelial lesion or malignancy(NILM).Overall,421(2.05%;95%CI,1.85–2.25)women were diagnosed with CIN1and above lesions,including 317(1.58%;95% CI,1.41–1.76)women with CIN1,93(0.50%;95% CI,0.37–0.56)women with CIN2/3,and 11(95% CI,27–99)with SCC.One hundred and four women(0.52%;95% CI,0.42–0.62)were diagnosed with CIN2 and above lesions(CIN2+).(2)HPV test results: 1036 cases of detection of high-risk human papilloma virus(HR-HPV)was positive in 293 cases,the total positive rate was 28.28%;the positive rate of CIN 1 HR-HPVfor HR-HPV25.87%,the positive rate of CIN2/3 group was 76.34%,the positive rates of control group 22.36%,each group compared with significant the difference(P< 0.001);the positive rate of HR-HPV showed a trend of increase with the aggravation of CIN lesions(P< 0.001);The most common HR-HPV genotypes were HPV16,HPV58 and HPV51.(3)the results of vaginal microflora detection: CIN 1,CIN2/3 group and control group,trichomonas vaginitis,vulvovaginal candidiasis,bacterial vaginosis and other types of vaginitis detection rate was no significant difference(P=0.079).(4)the results of dietary folic acid intake: The average daily dietaryfolate intake was significantly lower in the study group(344.61±153.07μg)than in the control group(371.50±166.58μg;P<0.001).(5)the analysis of risk factors of cervical intraepithelial neoplasia: univariate analysis,risk factors include: age,education,occupation,husband’s occupation,whether to clean the vulva after sex,whether to adopt contraceptive sterilization,sterilization time,HPV infection,HR-HPV infection and the average daily dietary intake of folate.The multivariate analysis results show that the above factors,including the risk factors of cervical intraepithelial neoplasia: high-risk HPV infection,56-65 years old,husband occupation for farmers,cleaning the vulva after sex,the average daily dietary folate intake less.Conclusion:(1)CIN and SCC detection rate are higher in Jiexiu area of Shanxi province;(2)the main risk factors of CIN including HPV infection,age 56-65 years old,husband occupation for farmers,cleaning the vulva after sex and the dietary folate intake less.PartⅡ Effects and mechanism of miR-224 targeting Smad4 on the biological function in cervical cancer cellsObjective: Cervical cancer is one of the most common gynecological malignant tumors in the world.Micro-RNAs(miRNAs)is involved in the proliferation,differentiation,apoptosis,invasion and metastasis of tumor cells,and plays an important role in the development of tumor.Mi R-224 as a member of the micro-RNAs family,miR-224 targeted to Smad4,but miR-224 targeting Smad4 has not been reported in cervical cancer.In this study,we study the effect of miR-224 targeting Smad4,and further clarify the effects and mechanism of miR-224 on the biological function of cervical cancer cells by regulating Smad4.Methods: We predicted the target gene of miR-224 by predicting the target gene on network.The expression of miR-224 and Smad4 was detected by quantitative real-time PCR.In order to clarify the miR-224 in cervical cancer targeting Smad4,in cervical cancer cell lines Si Ha and He La,using cell transfection technology over expression or silencing miR-224,q RT-PCR assay was used to detect the effect of miR-224 transfection.miR-224,CCK-8 cells and flow cytometry were used to detect biological behavior of Si Ha and Hela cells: cell proliferation,apoptosis,migration.The Smad4 protein was detected by Western blot in miR-224 overexpressing or silencing in Si Ha and He La cells,and then the effect of miR-224 on the regulation of cervical cancer cells was investigated by Smad4.Results: According to the target gene by bioinformatics prediction site,Smad4 was predicted to be a target of miR-224,miR-224 can bind to m RNA 3’-UTR of Smad4.InSi Ha and He La cervical cancer cells,compared with normal cells,the expression of miR-224 was significantly increased and the expression of Smad4 RNA was decreased.Overexpression of miR-224 can promote the proliferation and migration of Si Ha and He La cells,inhibit the apoptosis of cells,while silencing miR-224 can inhibit the proliferation and apoptosis of Si Ha and He La cells,and inhibit cell migration.The relationship between miR-224 and Smad4 in cervical cancer is that overexpression miR-224 expression was significantly decreased the expression of Smad4;while miR-224 was silent,Smad4 expression increased.Conclusion: The expression of miR-224 and Smad4 were closely related to cervical cancer cell procession.In Si Ha and He La cells,miR-224 overexpression can promote cell proliferation and migration,inhibit cell apoptosis,while silence for miR-224 can inhibit cell proliferation and migration,promote cell apoptosis.Overexpression of miR-224 could down-regulate the expression of Smad4,and the expression of Smad4 was up-regulated by silencing miR-224 expression.PartⅢ Effect of folate and miR-224 regulating autophagyin cervical cancer cellsObjective: Autophagy is a unique way of cell death.Autophagy plays an important role in tumor formation.Based on the autophagy of damaged organelles or abnormal material aggregation clearance to adjust the cell itself smoothly so as to stabilize the genes,suppressor cells into cancer cells,thereby reducing cell cancerization.However,the relationship between folate,cervical cancer and autophagy has not been studied.Thispaper studies the effects of autophagy related genes and miR-224,the relation between folate and Smad4.Methods: The expression of miR-224 and Smad4 was detected by quantitative real-time PCR.At the same time,the expression of Smad4 in cervical carcinoma was detected.In cervical cancer cell line Si Ha and He La,the expression of miR-224 was detected by quantitative real-time PCR and the effect of miR-224 transfection was verified by quantitative real-time PCR.Western blot was used to verify the expression of autophagy related protein,under the expression of overexpression or sclience miR-224 in Si Ha and He La cells.We study the relationship between folate and autophagy related genes.The expression of autophagy related genes Beclin1,LC3 and p62 and Smad4 m RNA and proteins in miR-224 overexpressing and silenced of Si Ha and He La cells were detected by quantitative real-time PCR and Westen blot methods.After the cells were treated with different concentrations of folic acid,those genes also were detected by quantitative real-time PCR and Western Blot.Results: Compared with normal cells,miR-224 was significantly increased and the expression of Smad4 m RNA was decreased in cervical cancer cell line.The expression of Smad4 in tissues of cervical intraepithelial neoplasia and cervical carcinoma was lower than that in normal tissues.Mi R-224 mimic and miR-224 inhibitor were successfully transfected into Si Ha and He La cells.Mi R-224 mimic can reduce the expression of Beclin1,LC3,Smad4,increase p62 expression.Si Ha and He La cells were transfected with miR-224 inhibitor,it could increase the expression of Beclin1,LC3 and Smad4,but decreased the expression of p62.A certain dose of folic acid can increase the expression of Beclin1,LC3 and Smad4 and decrease the expression of p62 in Si Ha and He La cells.Using different concentrations of folic acid interfere with Si Ha and He La cells.We found that both at the MRNA level and at the protein level,Beclin1,LC3,Smad4 expression were increased,p62 expression wasdecreased.Conclusion: miR-224 can affect the level of autophagy in cervical cancer cells by Smad4.Folic acid also affects the autophagy of cervical cancer cells.