The Expression Of ARHI In Cervical Carcinoma And The Regulatory Effect Of Resveratrol On That

Backgrounds and Objectives : Cervical cancer is now the most common gynecological malignancy of the world,which ranked second in the cause of female cancer related death.Its incidence is second only to breast cancer.It is one of the main causes of death in women from the developing countries.And more seriously,the incidence of cervical cancer in the world have obvious rising trend.And age tends to be younger,so the disease has been highly valued by scholars both at home and abroad.Up to now,most patients still choose surgery to treat cervical cancer,but the disease is prone to metastasis and recurrence.Now the treatment plan is to take the method of surgery combined with chemotherapy,but the chemotherapeutic drugs has a strong side effects and drug resistance phenomenon is also occur from time to time,seriously affected the quality of life of patients.To sum up,to find a more reliable and less toxic side effects of drugs,has a very important clinical significance and value.Resveratrol widely exists in plants and herbs of polyphenolic compounds,such as peanut,grape skin etc,and in many aspects it plays a beneficial biological effects,including anti-inflammatory,antioxidant,cardiovascular protection,prevention and inhibition of tumor.Previous studies have found that resveratrol can induce apoptosis of leukemia,liver cancer,breast cancer,ovarian cancer,prostate cancer,laryngeal cancer and other tumor cells.Thus,in tumor prevention and treatment on resveratrol has aimportant medicinal value,so the exploration discussing resveratrol in cervical cancer in regulating role is particularly important.ARHI gene is a tumor related gene cloned from human ovarian and breast epithelial cells and cancer cells.This gene contains three CpG islands,and its aberrant methylation has an effect on the expression of the gene.AHRI gene is an imprinted gene,normal cells,the parent source of ARHI allele occurred CpG island methylation,make its transcriptional activity was inhibited,and the paternal allele no methyl modification,which can be expressed in the progeny.Beclin is a homologous gene of yeast ATG6/Vps30 in mammalian cells,which plays an important role in the formation of autophagy.MAPLC3 is a homolog of mammalian cells in yeast Atg8 genes,positioning in autophagosome membranes,participate in the formation of autophagosomes,specific reflect the cell number of autophagosomes,has as the specific marker protein of autophagosomes,is considered more specific to be the autophagy marker for the diagnosis.Previous studies have also shown that the expression of ARHI is downregulated in many cancers,including breast carcinomas,ovarian cancers,pancreatic cancer and a few other cancers.But the research of ARHI in cervical cancer cell lines and tissues expression were rare.This study was conducted to evaluate the role of ARHI in human cervical cancers.We examined expression of ARHI in human cervical cancers and normal cervical tissue and also in 3 different cervical cancer cell lines to evaluate ARHI expression change with and without resveratrol treatment,and to analyze the correlation between this change and autophagy.Our data highlight the importance of ARHI in cervical cancer progression and provide the first biological basis for ARHI as a novel candidate target for gene therapy of cervical cancers,and provide more evidence for the value of resveratrol in clinical application.Materials and Methods: Paraffin specimens of 81 cases of human cervical cancer and normal cervical tissues were provided by Shengjing Hospital affiliated to China Medical University.Cervical adenocarcinoma cell line HeLa and cervical squamous cell carcinoma cell line SiHa and C33 A were purchased from the Chinese Academy of Sciences Shanghai cell bank.This experimental study based on paraffin embeddedtissue microarray technology,using immunohistochemical staining and trypan blue staining,immunocytochemistry,immunofluorescence,RT-PCR,Western blot analysis method to analyze the expression of the three cervical cancer cell lines related gene and protein with and without resveratrol treatment.Experimental data uses statistical software for statistical analysis.Results: 1)ARHI was frequently and highly expressed in SCCs(85.7%)and ACs(81.3%)in comparison with that of noncancersous squamous(44.4%)and glandular epithelia(68.4%),ARHI expression rate was 16.7% in rat cervix(p<0.01);2)HPV16exprssion was positive correlation with SE(r=0.509)and SC(r=0.440)group,Broad-spectrum HPV was positive correlation with SC(r=0.441)、GE(r=0.754)and AC group(r=0.521);3)After 48 hours of He La,SiHa and C33 A cells were treated with resveratrol and cisplatin,the cell number decreased,cell morphology changed,and the growth inhibitory effects of resveratrol and cisplatin were almost unanimous in cervical cancer cell lines(p>0.05);4)Using immunocytochemistry and immunofluorescence to detect the expression of ARHI protein and it was down regulated in the three cell lines HeLa,SiHa and C33 a with and without resveratrol treatment,that results are confirmed by western blotting and RT-PCR;5)The results of immunofluorescent staining and immunocytochemical staining also showed that the autophagy related protein Beclin and LC3 were up-regulated in the cervical cancer cells after resveratrol treatment;6)The test results by western-blotting showed that resveratrol could promote the expression of LC3 and also switching LC3 I to LC3 II.Conclusions: 1)Resveratrol has a similar inhibitory effect compared to the first line anti cervical cancer chemotherapy drug cisplatin,because it has no toxic side effect,it has more clinical application advantages than cisplatin.2)There were obvious apoptosis and autophagy in the sensitive cervical cancer cells,which showed that the improvement of autophagy activity could not prevent apoptosis or promote apoptosis;3)The positive rate and expression of ARHI in cervical carcinoma were significantly higher than that in normal cervical tissues,but the expression was down regulated after resveratrol treatment,this phenomenon suggests that the expression of ARHI with thehappening of cervical cancer is not a simple cause and effect relationship,HPV infection as a possible regulatory factor should be considered and analyzed.