MiR-218 Expression In Plasma Of Human Cervical Cancer And Its Effect On Enografts In Vivo

Cervical cancer has the highest incidence of the female reproductive system malignant tumor worldwide,which is a serious threat to women's health.Although persistent infection of high risk human papillomavirus(HPV)has been identified as a necessity for the occurrence of CC,a single HPV infection is not enough likely to induce CC since genetic and immune factors also take the play.MicroRNAs are endogenous noncoding RNA molecules.By binding to the 3 'end of its target genes,miRNAs cause degradation of the mRNA or inhibition of protein translation then alter the cellular and biological phenotype.It has shown that miR-218 is thought to be a potential tumor suppressor and has low expression in various of cancers,meantime it closely relates to tumor invasion and migration.Our previous study has found that miR-218 was lowly expressed in cervical cancer tissues and overexpression of miR-218 significantly inhibited invasion and migration of cervical cancer cells.The aim of this study was to detect the expression of miR-218 in the plasma of cervical cancer patients and to analyze the relationship between the expression of miR-218 and the clinicopathological features of cervical cancer patients.To observe the effect of miR-218 on the growth of cervical cancer cells in vivo and to explore its possible mechanism.Methods1.60 cases of cervical cancer and 60 cases of healthy persons(control group)were collected from January,2015 to June,2016 in the gynecology department of Guangxi Zhuang Autonomous Region People's Hospital.The expression level of miR-218 in cervical cancer tissues by qRT-PCR has been detected and the relationship between miR-218 expression and clinicopathological features in patients has been analyzed.2.MiR-218 and negative control(NC)lentivirus expression vector were used to infect cervical cancer HeLa and SiHa cells.The miR-218 cell line stably expressing miR-218 was screened and the fluorescence expression was observed under fluorescence microscope.The number of luminescent cells was detected by flow cytometry,while the expression of miR-218,miR-9,miR-21 and miR-31 in transfected HeLa and SiHa cells were detected by qRT-PCR.3.36 5-week-old female BALB/c nude mice were randomly divided into 6 groups(n = 6).The transplanted tumor model was established by injecting miR-218,miR-218-NC and control group cells suspension(1×107/L)into the axilla of the left forelimb of nude mice.The tumor diameter was measured every 3 days after tumor formation.And tumor volume was calculated(Tumor volume = 0.5 x long diameter x short axis 2),tumor growth curve was drawn.4 weeks later,the mice were sacrificed.The tumor was completely stripped and the tumor final weight and volume were measured.4.Total RNA was extracted from the tumor tissues were used Trizol and then Reverse transcriptase.The expression of miR-218,Rictor and mTOR mRNA were detected by qRT-PCR.The total protein in tumor tissues of each group was extracted,and the expression of Rictor and mTOR protein were detected by Western blot.B-actin was used as a control and Image J software was used to analyzes the strip gray values.Results1.The relative expression of miR-218 in plasma of patients with cervical cancer was significantly lower than that of normal controls(0.27 ± 0.14 compared to 1,P<0.05).The expression of miR-218 was lower in lymph node positive group(0.11 ±0.09)than in negative group(0.34 ± 0.11)P<0.05.And there was no significant difference in low-grade group,poorly differentiated group,vascular infiltration-negative group and tumor diameter>4cm group,P>0.05.2.The miR-218 lentiviral expression vector was successfully constructed.The expression of miR-218 in HeLa and SiHa cells after transfection was significantly higher than that in the control group,while the expression of miR-9,miR-21 and miR-31 had no significant changes.And flow cytometry showed that the fluorescence efficiency was>90%.3.The nude mice in each group had normal growth status.After 1 week,all nude mice showed subcutaneous tumor.The final volume and weight of miR-218 overexpressing group was significantly smaller than that of miR-218-NC and Control group whose difference was statistically significant(P<0.05).There was no significant difference in tumor volume and tumor weight between the control group and the control group.4.The relative expression of miR-218 in the miR-218 overexpressing group was significantly higher than that in the control group(1.053 ± 0.43 compared to 1.000 ±0.000)(P<0.05),while there was no statistically significant in the control group(P>0.05).The relative expression of Rictor and mTOR mRNA and protein in miR-218 groupwas significantly lower than that in control group,the difference was statistically significant(P<0.05).There was no significant difference in control group.P>0.05.Conclusions1.MiR-218 is down-regulated in plasma of cervical cancer patients and is closely related to the lymph node metastasis in patients.2.MiR-218 can inhibit the growth of transplanted tumor in nude mice.3.MiR-218 may be involved in the carcinogenesis and development of cervical cancer by inhibiting Rictor and regulating mTOR pathway.