Selenium Compound Inducing The Apoptosis Of Cervical Cancer Cells Through Regulating Apoptosis-related MicroRNA

AIM: To investigate the inducing effects of selenium dioxide (SeO2) on theapoptosis in human cervical carcinoma cell lines and its epigenetic role, which will behelpful in identifying the mechanisms of anti-cancer effects by selenium compound,and provide more experimental evidence to explore selenium as anti-cancer treatmentreagent.METHODS:1. Cell culture and sample treatment: Hela (HPV-18-positive) and Caski (HPV-16-positive) cells were selected to be the test subjects. They were cultured in RPMI1640medium supplied with10%FBS, and incubated at37C in humidified5%CO2-incubator. During exponential growth phase, cell lines were both divided into two groups randomly: experimental group treated with different concentrations of SeO2(1.875μmol/L,3.75μmol/L,7.5μmol/L,15μmol/L and30μmol/L for24h respectively) and control group (treated without SeO2for24h) in vitro; The samples were then collected for different assays accordingly.2. Content and methods: Morphological changes of Hela and Caski cells wereobserved by inverted optical microscope; Cell proliferation and activity wereexamined by MTT assay; Flow cytometry was employed to detect the cell apoptosis;The expressions of apoptosis-related proteins Caspase-3and P53in cervicalcarcinoma cell lines were determined by Western blot analysis; The effects of SeO2on apoptosis-related miRNA let-7a expression was detected by Stem-loop reversetranscription-polymerase chain reaction (RT-PCR).RESULTS:1. Morphological changes: observed with an inverted optical microscope, cell morphology was changed obviously in vitro. Compared with control group, cells became rounded and shrunken, granule in cytoplasm of experimental groups accumulated, normal morphology disappeared; The adherence cell number and the density of cells was evidently decreased while intercellular space enlarged dose-dependently with the treatment of SeO2. 2. Cell proliferation and activity: the cell proliferation inhibition was revealed byMTT assay, which showed that SeO2markedly inhibited cervical cell linesproliferation and viability in a dose-dependent manner; The inhibitory rates of Helacells treated with1.875μmol/L,3.75μmol/L,7.5μmol/L,15μmol/L,30μmol/L SeO2were1.22%,20.25%,35.03%,43.56%,60.74%, respectively. Among them, theinhibitory effects induced by7.5μmol/L,15μmol/L and30μmol/L of SeO2aresignificant（P＜0.05） compared with the control group; The inhibitory effects onCaski is rising as the concentrations increased, the inhibitory rates of Caski cellstreated with1.875μmol/L,3.75μmol/L,7.5μmol/L,15μmol/L,30μmol/L SeO2were14.39%,34.08%,43.01%,57.73%,68.65%respectively, with statistical significance（P＜0.05） totally compared with the control group.3. Apoptosis rate: Flow cytometric analysis demonstrated that the apoptosis ratesinduced by SeO2increased dose-dependently in cervical carcinoma cell lines. Theapoptosis rates of Hela cells treated with0μmol/L,1.875μmol/L,3.75μmol/L,7.5μmol/L,15μmol/L,30μmol/L were3.12%,30.56%,33.42%,37.50%,45.43%,69.38%, respectively; The apoptosis rates of Caski cells treated with0μmol/L,1.875μmol/L,3.75μmol/L,7.5μmol/L,15μmol/L,30μmol/L were3.7%,10.7%,67.5%,78.2%,87.1%,85.2%, respectively.4. Caspase-3and P53expressions: Western blot analysis showed that SeO2could up-regulate the apoptosis-related proteins levels in cervical carcinoma cell lines.The expressions of Caspase-3and P53in Hela cells all have significant differencescompared with control group (P＜0.05), and peaked at the concentration of7.5μmol/L. Treated with lower concentrations of SeO2, the expression of Caspase-3on Caski cells had a minor change, while with highconcentrations(7.5μmol/L,15μmol/L,30μmol/L), changes are significant (P＜0.05).Meanwhile, as the SeO2concentrations increased, the induce effects of P53on Caskicells significantly raised compared with control group (P＜0.05).5. miRNA let-7a levels: results from Step-loop Realtime PCR demonstrated thatcompared with the control groups, SeO2could induce the expression ofapoptosis-related miRNA let-7a both in Hela cells and Caski cells with statisticalmeanings (P＜0.05); the induction reached its climax at the concentration of 7.5μmol/L bothly, the values of-⊿⊿Ct were up to7.79in Hela cells and14.06inCaski cells respectively.CONCLUSION: SeO2showed anti-tumor properties via apoptosis pathwayby up-regulating the expressions of apoptosis-related proteins Caspase-3， themechanisms involved inducing effects of P53and let-7a in cervical cancer cell lines.