| The solid tumor hypoxia has become a central issue in cancer treatment, whichactivates serials of hypoxia response genes contributing to tumor adapts hypoxicenvironment. The gene of hypoxia-inducible factor1(HIF-1) plays a central role fortumor adapting hypoxic environment by activating transcription of many genes,including those involved in angiogenesis, energy metabolism, tumor invasion andmetastasis, and closely associated with radiotherapy and chemotherapy sensitivity.The two subunits of HIF-1α and β, subunit α as the main function structure unit. Byradiotherapy, surround cancer cells easy killed, while there is radio-resistance in thecentral of the solid tumors because the hypoxic environment. Autophagy is aself-digestive process to degradation of intracellular unfolded protein and damagedorganelles, thus maintains cells homeostasis. However, autophagy can behave as atumor suppressor or oncogene. The similar paradox is exhibited during tumor therapy,in which autophagy could play pro-survival role or leads to programmed cell death.The exact roles of HIF-1α are sill largely unknown in autophagy process andespecially in radiation-induced autophagy.Objective: To explore the impact of HIF-1α on autophagy process and identifythe effects of HIF-1α in radiation therapy in breast cancer.Methods:(1) Human breast cancer cell line MCF7was used and differenttreatment groups, including sham-rdiation group(sam), hypoxia group and hypoxiacombined with radiation group, were demonstrated during the experiment.(2)150μmol/L CoC12was used to treat MCF-7cells for simulating hypoxia environment.(3)shRNA oligonucleotide retrovirus vector was constrcted targeting to HIF-1alpha gene,calcium phosphate transfection method was used to create a MCF-7HIF-1RNAimodel.(4) MDC staining and Hoechst staining were used to monitor autophagy andapoptosis, respectively.(5) Colony assay was used to analysis the radiation sensitivity.(6) Western blot was used to analyze protein expression.Results:1The activation effect of hypoxia on HIF-1α expression and autophagy processWestern blot was used to detect HIF-1α expression of different treatment groupsof MCF-7cells. The expression of HIF-1α protein could not be detected insham-control and radiation groups. However, the expression of HIF-1α proteinincreased obviously in hypoxia and radiation groups. The expression of HIF-1αfurther increased in the hypoxia combined with radiation group. Quantity Onesoftware was use to evaluate the band value of HIF-1α and MAPLC3protein. In thesham-control, radiation, hypoxia and hypoxia combined with radiation group, thevalue of HIF-1α protein was0,0,1and1.89, the value of LC3II/LC3I was1.15,1.73,2.38and3.60, respectively.MDC staining was used to further monitor the autophagy process. The autophagypositive cells were7%in control group. While the autophagy positive cells increasedto15%and18%(p<0.05), and hypoxia combined with radiation group increased to23%(p<0.05). These results indicated that hypoxia induced the expression of HIF-1αin MCF-7cells, both hypoxia and radiation induced autophagy, and hypoxiacombined with radiation further activated autophagy.2Establishment of HIF-1α gene silencing modelOligonucleotide shRNA(small hair RNA) was designed and synthesized bybioinformatics targeting human HIF-1α gene, restriction endonuclease of BglII andHindIII were designed at both ends respectively. After annealing and phosphorylation,double-stranded shRNA were connected with pSUPER vector to form pSUPER-HIF-1α Ri expression vector. pSUPER-HIF-1α Ri or pSUPER vector was transfectedinto MCF-7cells, western blot was used to evaluate the expression of HIF-1α.48hafter the treatment of MCF-7cells with CoC12,the expression of HIF-1α wasdetectable in MCF-7-pSUPER cells, while the expression of HIF-1α decreased inMCF-7HIF-1α Ri cells, and not detectable in radiation group of MCF-7-pSUPERcells. The expression of HIF-1α in hypoxia and hypoxia combined with radiationgroups was detectable in MCF-7-pSUPER cells, especially in hypoxia combined withradiation. While weakly expressed in hypoxia and hypoxia combined with radiationgroups of MCF-7-HIF-1α Ri cells.3The enhancement effect of HIF-1α in hypoxia and radiation-inducedautophagyMDC staining was used to monitor autophagy. In sham-control group, radiation group, hypoxia group and hypoxia combined with radiation group the positiveautophagy of MCF-7-pSUPER and MCF-7-HIF-1α Ri cells were6%,6%,14%,11%,and17.5%,12.5%,20%,15%(p<0.05), respectively. These results indicated thatsilencing of HIF-1α inhibited autophagy induced by hypoxia and radiation.To further explore the relationship of HIF-1α and autophagy, western blot wasused to evaluate the expression of autophagy-related protein MAPLC3. In sham-control group, radiation group, hypoxia group and hypoxia combined with radiationgroup of MCF-7-pSUPER cells, the value of LC3II/GAPDH were1,1.5,2.2,2.3.While in same treatment groups in MCF-7-HIF-1α Ri cells the value ofLC3II/GAPDH were0.84,0.92,0.82,1.1. These results indicated that silencing ofHIF-1α could inhibit the hypoxia and radiation induced autophagy in MCF-7cells.4The silencing of HIF-1α enhanced the radiation sensitivity of MCF-7cellsunder hypoxia environment.Colony assay was used to analysis cells survival fraction. Compared with controlgroup, the survival fraction of MCF-7-pSUPER cells increased in different hypoxiacombined with radiation groups(2,4,6and8Gy, p<0.05). Compared with controlgroup, for survival fraction of MCF-7-HIF-1α Ri cells there is no statisticallysignificantly in combined with radiation groups(2,4,6and8Gy, p<0.05). Theseresults indicated that the silencing of HIF-1α enhanced the radiation sensitivity ofMCF-7cells which suffering hypoxia environment.5The inhibition of autophagy enhanced radiation sensitivity of MCF-7cellsunder hypoxia environment.3MA was used to inhibit autophagy, colony assay was used to analysis cellssurvival fraction. Compared with control group, after3MA treatment the survivalfraction of MCF-7cells decreased(p<0.05), suggesting3MA increased radiationsensitivity. While the survival fraction increased in hypoxia group when MCF-7cellssuffering hypoxia environment in different radiation dose treatment(p<0.05),suggesting hypoxia decreased radiation sensitivity. The survival fraction of hypoxiacombined with3MA treatment group was between hypoxia group and controlgroup(p<0.05), suggesting3MA could reverse the radiation resistance induced byhypoxia. These results indicated that hypoxia induces radiation resistance in MCF-7cell, at least partially attributed to the activation of autophagy by HIF-1α expression.6The mechanism of HIF-1α in radiation-induced autophagy MCF-7-pSUPER and MCF-7-HIF-1α Ri cells were divided into sham-controlgroup, radiation group, hypoxia group and hypoxia combined with radiation group,respectively. Western blot was used to detect the expression of AKT, mTOR, p70. InMCF-7-pSUPER cells, the AKT/GAPDH value of each group was1,0.61,0.55,0.41,the mTOR/GAPDH value of each group was1,0.7,0.51,0.2, the p70/GAPDH valueof each group was1,0.5,0.3,0.2, suggesting that radiation, hypoxia, hypoxiacombined with radiation decreased the expression of AKT, mTOR, p70, especially inhypoxia combined with radiation group. In MCF-7-HIF-1α Ri cells, the AKT/GAPDHvalue of each group was1.1,1.21,0.76,1.38, the mTOR/GAPDH value of each groupwas0.85,0.79,0.88,0.93, the p70/GAPDH value of each group was0.86,0.62,0.8,0.62. Compared with the same treatment group of MCF-7-pSUPER cells, theexpression of AKT, mTOR, p70increased in MCF-7-HIF-1α Ri cells. These resultsindicated that HIF-1α increased radiation-induced autophagy in human breast cancerMCF-7cells under hypoxia environment, which might be AKT/mTOR pathway–dependent.7The silencing of HIF-1α has no effect on apoptosis after the treatment ofhypoxia and radiationPI staining was used to monitor cells apoptosis. In both MCF-7-pSUPER andMCF-7-HIF-1α Ri cells, as compared with control group, the apoptosis positive cellsincreased in radiation, hypoxia, hypoxia combined with radiation group. There was nostatistically significant between MCF-7-pSUPER and MCF-7-pSUPER-HIF-1α Ricells in the same treatment group. The apoptosis-related protein PARP was alsodetected. In MCF-7-pSUPER cells, the cleaved PARP/PARP values of control,radiation, hypoxia, hypoxia combined with radiation group were1,2.07,1.07,0.89,respectively. In MCF-7-HIF-1α Ri cells, the cleaved PARP/PARP values of control,radiation, hypoxia, hypoxia combined with radiation group were0.84,1.97,0.76,0.86,respectively. There was no statistically significant of the cleaved PARP/PARP valuesbetween MCF-7-pSUPER and MCF-7-HIF-1α Ri cells in the same treatment group.Conclusion1. Hypoxia activates the expression of HIF-1α and autophagy process.2. The silencing of HIF-1α enhanced the radiation sensitivity of human breastcancer MCF-7cells which suffering hypoxia environment.3. Hypoxia environment increased radiation resistance in MCF-7cell, partially attributing to the activation of autophagy by HIF-1α expression.4. HIF-1α increased radiation-induced autophagy in human breast cancer MCF-7cells under hypoxia environment, which might be AKT/mTOR pathway dependent.5. he silencing of HIF-1α does not affect hypoxia and radiation-inducedapoptosis in MCF-7cells... |
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