Studies On The Roles Of Mir-21in Radiation-induced Autophagy In Cervical Cancer

Autophagy is a conserved process amongst eukaryotes for degradating andrecycling, by which the unwanted and damaged cellular components includingproteins and organells in response to diverse stress are eliminated. Autophagy playsan important role in cancer. On one hand, autophagy, also named astypeⅡ programmed cell death,functions as tumor suppressors. On the other hand,autophagy enhances tumorigenesis and protects tumor cells from cell death.MicroRNAs, the short non-coding RNAs, with the combination of the targetgene3’-UTR, they induce mRNA degradation or transcriptional suppression, theninhibiting the synthesis of the target protein and regulating cell proliferation,apoptosisand autophagy. In recent years, several studies have confirmed that miR-21regulates cancer gene in breast cancer,gliblastoma,prostate cancer and colon cancer.Radiation therapy is a routine treatment of cervical cancer, about80%of cericalcancer patients received single radiation or comprehensive therapy. miR-21has a highexpression level in cervical cancer cells. Functions and the target genes of miR-21arelargely unknown especially in autophagy process and autophagy induced by ionizingradiation (IR) treatment.ObjectiveTo explore the effect of miR-21on the process of autophagy and identify therelated target genes in human cervical cancer cells. The effect of miR-21after IRtreatment in cervical cancer will be also demonstrated.Methods(1) Human cervical cancer cell line HeLa and SiHa were used in this study;(2)X-irradiation was used and irradiation conditions were as follows: voltage180kV,current18.0mA, filtration plate0.25mm Cu and1.0mm Al, distance between targetand X source60cm, dose rate0.344Gy/min;(3) qRT-PCR was used todetectendogenous miRNA expression;(4)Western blot was used to analyze proteinexpression;(5) Cells viability was evaluated by MTT method;(6) Cell apoptosis and autophagy were analyzed by FACS assay;(7) Calcium phosphate transfection methodwas used to create a HeLa-Beclin1RNAi model.(8) Student t-test and χ2test wereused for statistical purpose;(9) Quantity One software was used to calculate the bandvalue.Results1. IR induced cell autophagy and increased miR-21levelThe ratio of MAPLC3Ⅱ/LC3Ⅰwas higher than control group at16h,24h and32h after IR treatment in HeLa and SiHa cells. By qRT-PCR analysis, miR-21levelincreased to1.7folds and1.15folds after IR treatment in HeLa and SiHacells,respectively.2. miR-21regulated the expression of autophagy related gene Beclin1The Beclin1expression increased after IR treatment compared with sham-IRgroup in HeLa cells. Western blot results showed that the expression of Beclin1increased obviously in mimic group(compare with NC group) and mimic+4Gygroup(compare with NC+4Gy group).While in SiHa cells, western blot resultsshowed that the expression of Beclin1increased after IR treatment compared withsham-IR group in SiHa cells.The expression of Beclin1was lower in mimic groupcompared with NC group. The expression of Beclin1was lower in mimic+4Gy groupcompared with NC+4Gy group.3. Effects of miR-21on radiation induced cell death in HeLa cells①Effects of miR-21on radiation induced cell viability: HeLa cells transfectedwith miR-21NC、mimics、inhibitor NC or inhibitor. Add different doses of IRtreatment(0,4Gy,8Gy)24h after transfection. MTT assary showed that there were nochanges of cells viability in HeLa cells.②Effects of miR-21on radiation inducedcell apoptosis: After transfection with miR-21mimics, compared with control group,radiation induced apoptosis had little increased. Compare with NC+4Gy group,apoptosis had significantly increased in mimic+4Gy group(P<0.05). The apoptosislevel in inhibitor group was lower than inhibitor NC group. But there was no changein the apoptosis level between inhibitor+4Gy group and inhibitor NC+4Gy group.③Effects of miR-21on radiation induced cell autophagy: After transfection withmiR-21mimics, compared with control group, radiation induced autophagy hadincreased (P<0.05). The autophagy level in mimic+4Gy group was higher than NC +4Gy group(P<0.05). These results indicate that miR-21enhance IR-inducedapoptosis and autophagy, but have no effect on cell viability in HeLa cells.4. Effects of miR-21on radiation induced cell death in SiHa cells①Effects of miR-21on radiation induced cell viability:the cell viabilitywassignificantly increased in mimic+8Gy group compared with NC+8Gy group.Thisresult indicated that miR-21inhibited IR-induced cell death in SiHa cells.②Effectsof miR-21on radiation induced cell apoptosis: Compare with NC+4Gy group,apoptosis had significantly decreased in mimic+4Gy group(P<0.05).③Effects ofmiR-21on radiation induced cell autophagy: the autophagy level in mimic+4Gygroup was lower than NC+4Gy group(P<0.05). Compared with inhibitor NC+4Gygroup, autophagy had significantly increased in inhibitor+4Gy group(P<0.05). Theseresults indicate that miR-21inhibit IR-induced autophagy in SiHa cells and viceversa.Conclusion1. miR-21regulates autophagy process in HeLa and SiHa cervical cancer celllines, but appears different outcomes. miR-21enhances IR-induced autophagy inHeLa cells, but suppresses IR-induced autophagy in SiHa cells.2. miR-21increases the expression of Beclin1in HeLa cells, suppresses theexpression of Beclin1in SiHa cells.3. miR-21inhibits IR-induced cell death in SiHa cells.