| BACKGROUND AND OBJECTIVEHepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies in the world. The mortality rate was the third among the world’s malignant tumor death. China is a high incidence area of liver cancer, The morbidity and mortality was the second place in malignant tumor, which accounts for 50% deaths of patients with liver cancer. Treatment for HCC including surgical resection, liver transplantation, radiation therapy, interventional therapy, minimally invasive ablation and targeted therapy and so on. however, those with HCC are only eligible for a transplant during the early stages of this disease. Surgical resection provides the best chance for a cure in select patients. Unfortunately,80% patients was diagnosed in the late stage and lost the operation opportunity. surgery is often followed by a high rate of tumor recurrence, which is often the main cause of long-term treatment failure. The 5-year rate of HCC recurrence following resection has been reported as high as 70%.Radiation therapy (RT) is used in approximately one-third of all patients with cancer. The development of a three-dimensional conformal technique (3D-CRT) and intensity-modulated radiotherapy (IMRT) techniques allows the delivery of higher doses of radiation to the target tissue, thereby sparing the majority of the liver from radiation damage. In a series of retrospective and prospective studies, it was determined that RT is a safe and effective local therapy with potential benefits for patients who are unable to undergo surgery. There are certain limitations to the use of RT in patients with HCC, normal liver tissue is radiosensitive organs,75% of patients will suffer from liver dysfunction when the whole liver irradiation with accumulative doses of more than 40Gy, while the radiation lethal dose of hepatoma cells was at least 60Gy, significantly exceeded the tolerance dose of normal liver cells. Moreover, 80% patients with hepatocellular carcinoma in our county accompanying various degree of liver cirrhosis and reduce the ability of radiation tolerance. Therefore, how to improve the radiation sensitivity of HCC, is the key issue need to be solved, If cellular sensitivity to radiation can be enhanced, we may be able to improve the benefit of RT in HCC and reduce the toxicity of this approach. Therefore, how to improve the tumor radiosensitivity is a key problem need to be solved.PI3K/AKT signaling pathway plays an important role in tumorigenesis, development and survival process. Active AKT gains a variety of biological activities which including promoting tumor cell growth, proliferation, invasion and metastasis, inhibition of apoptosis, regulating endothelial growth, angiogenesis, and increasing sensitivity to radiation through a series of catalytic protein phosphorylation. PTEN gene (phosphatase and tensin homologdeleted from chromosome 10) is located on human chromosome 10q23.3, which is the only tumor suppressor gene with phosphatase activity in vivo. PTEN gene in human cells widely expressed in normal tissues. PTEN is widely expressed in human cells and normal tissues. Studies have shown that PTEN expression deletion or mutation in a variety of human malignant solid tumors (including glioblastoma, prostate cancer, melanoma, thyroid cancer and bladder cancer, etc.) and hereditary tumor predisposition syndrome. PTEN can regulate a series of intracellular protein phosphatase activity, resulting in deletion or mutation of the gene which plays an vital role in in tumor development. Kawamura and his collaborators found that the rate of PTEN deletion allele was 28.1% by detecting 89 cases of hepatocellular carcinoma tissues. Fujiwara and his collaborators found that rate of PTEN deletion allele was 32.4% by detecting 37 cases of HCC. As we know, the regulation of gene expression has multiple levels and post-transcriptional is also important as well as iitiation transcriptional regulation. microRNAs (miRNAs) are a class of non-coding small molecule RNA, about 17-25 nucleotides which was recently found. microRNAs could degrade mRNA or inhibit translation by binding to targeted gene non-coding region which at last achieve negative regulation of gene expression at the post-transcriptional level. microRNAs are closely related to the development of tissues and organs of animal and plant, cell differentiation, apoptosis, fat metabolism and other vital activities, abnormal expression of which will lead to the major diseases, such as cancer. Abnormal expression levels of miRNAs was existed in many tumors, which function both oncogenes and tumor suppressor genes. Recent studies have found miRNAs were closely related to tumorgenesis, metastasis, clinical treatment and prognosis.It has been found that miRNAs are closely associated with tumor radiosensitivity, such as Lin28-let7 reshapes the radiosensitivity of tumor through activating K-Ras, miR-9 and let-7g enhanced tumor radiosensitivity by inhibiting the expression of NFkappaBl, MicroRNA-181a enhance the radiosensitivity of malignant glioblastoma by targeting Bcl-2, miR-23b enhance cell radiosensitivity by inhibiting autophagy in breast cancer.MiR-20a was abnormally expressed in many common cancers and plays an important role in the tumorigenesis, development and metastases. However, the role of miR-20a in HCC radiationresistance is still unclear.The aim of this study was exploring the role of miR-20a in HCC radioresistance and provide important theoretical basis for clinical HCC radiation therapy.This study found that miR-20a could diminsh HCC cells radiosensitivity by inhibiting PTEN, and then activate the PI3K/AKT pathway which at last induces cells radioresistance in HCC. In this study, miR-20a study molecular mechanisms of liver cancer radiation sensitizing effect of providing new insights for miR-20a for the treatment of liver cancer provides a new theoretical basis for the radiotherapy of HCC.METHODS一. Detection of miR-20a and PTEN in HCC cell lines and tissues1. Detection of miR-20a in HCC cells and tissues by quantitative PCRThe normal human liver cell line LO2, the HCC cell lines Bel-7402, SMMC-7721, Bel-7402, QGY-7701 and HCCLM3 were used in this study. Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer’s protocol. The cDNA library was synthesized using the PrimeScript RT reagent kit following the manufacturer’s instruction. For mature miRNA quantification, cDNA was generated using specific stem-loop universal primers. RT-PCR for miR-20a and PTEN in these six cell lines was performed using SYBR Premix Ex Taq II according to the manufacturer’s protocol and was measured using an Agilent Stratagene Mx3005P Sequence Detection System. U6 and GAPDH was used as the internal control.The 28 cases of fresh hepatocellular carcinoma and matched adjacent tissues were stored in liquid nitrogen and extracted total RNA from the samples according to instructions. The cDNA was synthesized using the PrimeScript RT reagent kit following the manufacturer’s instruction. For mature miRNA quantification, cDNA was generated using specific stem-loop universal primers. RT-PCR for miR-20a and PTEN in 28 pairs of samples was performed using SYBR Premix Ex Taq II according to the manufacturer’s protocol and was measured using an Agilent Stratagene Mx3005P Sequence Detection System. U6 and GAPDH was used as the internal control.2. Detection of PTEN in HCC cells and tissues by quantitative PCR and westernblotThe normal human liver cell line LO2 and the HCC cell lines Bel-7402, SMMC-7721, Bel-7402, QGY-7701 and HCCLM3 were collected and total protein was extracted from the cells according to the manufacturer’s protocol. Protein concentrations were determined according to the BCA method.5×SDS-PAGE loading Buffer was added into the protein to denaturation, following electrophoresis, transmembrane, blocking, incubation for primary antibody and secondary antibody, Finally, ECL method is used to the development on the developing instrument to detect the expression levels of PTEN in these six cells.The protein of HCC samples were extracted according to the manufacturer’s protocol. The expression levels of PTEN in those 28 paired tissues was detected by quantitative PCR and westernblot.二. The effect of miR-20a on cell radiosensitivity in HCC1. Establishing of stable GFP-miR-20a overexpressing cellsThe recombinant lentivirus containing GFP-miR-20a was purchased from Genepharma (Shanghai, China). Bel-7402 and SMMC-7721 cells were infected with lentivirus particles and isolated via fluorescence-activated cell sorting to generate populations stably expressing GFP-miR-20a.2. miR-20a mimics and inhibitor transfectionThe miRNA mimics, the inhibitor, the control oligonucleotides were purchased from RiBoBio. Transfection of oligonucleotides and siRNA was performed using Lipofectamine 2000 reagent according to the manufacturer’s protocol.3. Irradiation and clonogenic survival assaysIncreasing numbers of cells were seeded on 6-well plates (previously transfected with oligonucleotides or virus for 48 hours) in triplicate and exposed to the indicated dose of radiation using 4MV X-rays generated from linear accelerators at a dose rate of 5 Gy/min. The cells were fixed using 100% methanol and stained using 1% crystal violet after incubation at 37℃ for 10 to 14 days. Colonies containing>50 cells were counted via microscopic inspection. The surviving fraction was calculated according to the multi-target single-hit model.4. Western blot analysisTotal cell lysates were extracted using radio-immunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitor cocktails and quantified using a bicinchoninic acid protein assay kit. Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked using 5% nonfat milk for 1 hour at room temperature, followed by incubation in the primary antibodies overnight at 4℃. After washing three times, the membranes were incubated in HRP-conjugated secondary antibodies for 1 hour at room temperature. The immunoreactive bands were visualized using an ECL western blot substrate.三. The molecular mechanisms of miR-20a on HCC cell radioresistance1. Bioinformatic analysis for predicting miR-20a target genesTo investigate the molecular mechanism by which miR-20a suppressed to induce cell radioresistance, we identified PTEN was a potential target of miR-20a.2. Luciferase reporter assayThe 3’untranslated region (UTR) of PTEN and mutant 3’UTR, which contains the putative miR-20a binding site, were amplified from genomic DNA via PCR and cloned into the Renilla luciferase gene (pLUC-REPORT vector, Promega, Madison, WI, USA). For the luciferase reporter assay, Bel-7402 and SMMC-7721 cells were co-transfected with a luciferase reporter vector (either pLUC-3’UTR-PTEN or pLUC-3’UTR-mut-PTEN) and negative control miRNA or the miR-20a mimic, inhibitor. Forty-eight hours after transfection, the cells were assayed for luciferase activity using the Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. For each sample, the relative luciferase activity was normalized to firefly luciferase activity. Three independent experiments were performed in triplicate.3. Analysis of the expression of PTEN mRNA and protein after ectopic expression of miR-20a in HCC cellsQuantitative real-time PCR (RT-PCR) and Western blot analysis were used to analyzed the expression of PTEN mRNA and protein after transfection with miR-20a mimics and inhibitor.4. The effect of PTEN knockdown on HCC cell radiosensitivityThe PTEN knockdown lentivirus was purchased from Genepharma (Shanghai, China). Bel-7402 and SMMC-7721 cells were infected with lentivirus particles and isolated via fluorescence-activated cell sorting to generate populations stably expressing GFP-PTEN. The surviving fraction was calculated according to the multi-target single-hit model.5. The effect of PTEN overexpression on HCC cell radiosensitivityThe transfection of pCDNA3.1-PTEN and control was performed using Lipofectamine 2000 reagent according to the manufacturer’s protocol. After transfection for 24h, cells were treated as indicated and then calculate colony formation efficiency and survival fraction according to the multi-target single-hit model.6. Restoration the expression of PTEN to identity whether it is a functional target of miR-20aTransfection of pCDNA3.1-PTEN and control into cells stably overexpressing miR-20a by using Lipofectamine 2000 reagent according to the manufacturer’s protocol. Then cells were treated as indicated. The surviving fraction was calculated according to the multi-target single-hit model.7. The effect of PI3K/AKT pathway inhibitor LY294002 on HCC cells radiosensitivityThe miRNA mimics, the inhibitor, the control oligonucleotides were transfected into cells by using Lipofectamine 2000 reagent according to the manufacturer’s protocol.24h later, cells were added LY294002 or not for 1h, then exposed to irradiation, The surviving fraction was calculated according to the multi-target single-hit model.8. Signaling pathway-related protein was analyzed by westernblotThe related protein levels such as PTEN, AKT and p-AKT were detected by westernblot when cells were treated as indicated.9. Animal studiesAll of the animal experiments were carried out in strict adherence with the Regulations for the Administration of Affairs Concerning Experimental Animals. All procedures involving animals and their care in this study were approved by the Institutional Animal Care and Use Committee. For xenograft tumor assays,1×106 cells as indicated were injected subcutaneously into the back of 4 to 6-week-old nude mice. After tumors were detected, the tumor size was measured every 3 during a slide caliper. When the tumor volume reached 200 mm3, it was irradiated with a single 10 Gy dose 10 days after the injection. Tumor size was calculated every two days using the following formula:(length×width2)/2. Tumor growth days was expressed in terms of time in days for irradiated xenografts to increase in volume from 200 mm3 to 1000 mm3. Westernblot analyzed the expression of PTEN and p-AKT after overexpression of miR-20a.10. Statistical analysisAll values are expressed as the mean±standard deviation. Results were analyzed by ANOVA or using a two-tailed Student’s t test. Survival analysis was carried out using the Kaplan-Meier method. SPSS 13.0 software (SPSS; North Chicago, IL, USA) was used for statistical analysis, with statistical significance established at.P<0.05. All Western blot results were quantified using Bio-Rad lab image quantitative analysis software. All data are presented as the mean±SD of results from three independent experiments.Results:1. Intracellular miR-20a levels in HCC tissue samples and cell lines increased compared with adjacent non-tumor tissue and were inversely correlated with PTEN levels.In 28 HCC tissue samples, we found that miR-20a levels were increased compared with matched adjacent non-cancerous tissues and HCC cell lines. By contrast, PTEN levels decreased in both HCC specimens and cell lines, as indicated by qRT-PCR and Western blot analysis.2. MiR-20a induces HCC cells resistance to radiation treatment.We investigated the effect of miR-20a on HCC cell radiosensitivity. Overexpression of miR-20a increased the survival fraction of Bel-7402 and SMMC-7721 cells and inhibition of miR-20a in HCCLM3 and QGY-7701 cells resulted in an increased survival fractions subjected to different doses of irradiation. In addition, an increase in miR-20a activity in the Bel-7402 and SMMC-7721 cells was followed by a decrease in PTEN and caspase-3 activity, as well as a decrease in phosphorylation of the histone H2AX(y-H2AX), an indicator of the cellular response to DNA damage. It also resulted in an increase in Bcl-2 and P-AKT levels in these cells. Correspondingly, a decrease in miR-20a activity achieved the opposite results. It was well documented that the capacity of anti-apoptosis of cancer cells is closely associated with radioresistance. We further measured IR-induced apoptosis in HCC cells overexpression of miR-20a. We found miR-20a overexpression significantly decreased cells apoptosis in HCC cells than the controls when combination with IR.3. PTEN is a target of miR-20a.We predicted that miR-20a targets the PTEN 3’-UTR region. Using TargetScan, we found a perfect base pairing between the seed sequence of miR-20a and the 3’-UTR of PTEN mRNA and that these seed sequences were conserved across species. We then cloned the target sequence of wild-type PTEN 3’-UTR (wt 3’-UTR) or the mutant sequence (mt 3’-UTR) into a luciferase reporter vector. HEK293 cells were then transfected with a wt or mt 3’-UTR vector and with miR-20a mimics. This resulted in a significant decrease in luciferase activity compared with miRNA controls. The mt 3’-UTR vector was not affected by simultaneous transfection with miR-20a mimics. Furthermore, cotransfection with the anti-miR-20a and wt 3’-UTR vector in HEK293 cells was followed by an increase in luciferase activity. Westernblot and qRT-PCR analysis indicated PTEN activity had an inverse correlation with miR-20a.4. miR-20a inhibits the expression of PTENEctopic expression of miR-20a significantly suppressed the mRNA and protein expression of PTEN. Moreover, knockdown of miR-20a could up-regulate the expression of PTEN.5. PTEN sensitized HCC cells to radiation treatment.To confirm that miR-20a enhanced radiosensitivity due to the direct targeting of PTEN, we examined the effect of silencing the expression of PTEN on radiosensitivity. We found that knocked-down PTEN could increase the survival fraction of Bel-7402 and SMMC-7721 cells when subjected to radiation (0-8Gy) accompanied by down-regulation of caspase-3 and y-H2AX expression and up-regulation of Bcl-2 and p-AKT expression, up-regulation of PTEN in HCCLM3 and QGY-7701 cells had an opposite effect. Apoptosis assay showed that PTEN silencing resulted in much less apoptosis than those for each treatment separately treated with radiation.6. PTEN reversed the effects of miR-20a on cell radioresistance.To determine whether the effect of miR-20a on cell radiosensitivity was mediated by the inhibition of PTEN expression, we transfected pcDNA3.1-PTEN into cells overexpressing miR-20a. Overexpression of PTEN decreased the survival fraction of transfected cells compared with non-transfected cells, which suggests that PTEN reverses the effects of miR-20a. Cell apoptosis suggested that ectopic expression of PTEN significantly rescued miR-20a-induced radioresistance enhancement. Western blot analysis indicated that PTEN may also restore the expression of caspase-3, y-H2AX and reduced Bcl-2 expression.7. miR-20a induces HCC cells radioresistance by activating PTEN/PI3K/AKT pathways.We found that overexpression of miR-20 reduced the expression of PTEN while simultaneously increasing the expression of P-AKT. AKT is the core component of the PTEN/PI3K signaling pathway, therefore, full inactivation of AKT could be the key to enhancing the radiosensitivity of tumor cells. On the basis of these findings, we investigated the ability of miR-20a to induce radioresistance by activating the PTEN/PI3K/AKT pathway. We found that exposure to the tyrosinekinase inhibitor LY294002 reverses the effect of miR-20a.8. miR-20a induces cell resistance to irradiation treatment in vivoTo determine whether miR-20a induces tumor resistance to the effects of radiation in vivo, we irradiated the tumor tissue using a single 10 Gy dose administered 10 days after injection. We found that the size of the xenografts derived from tissue overexpressing miR-20a was much greater than the size of xenografts derived from controls exposed to the same dose of radiation and resulted in tumor re-growth delay. Western blot analysis suggested that PTEN expression was reduced and the expression of p-AKT was up-regulated in xenografts treated as described.Conclusion:1. Intracellular miR-20a levels in HCC tissue samples and cell lines increased compared with adjacent non-tumor tissue and were inversely correlated with PTEN levels.2. MiR-20a induces HCC cells resistance to radiation treatment.3. PTEN sensitized HCC cells to radiation treatment.4. PTEN reversed the effects of miR-20a on cell radioresistance.5. PTEN is a direct functional target of miR-20a.6. miR-20a induces HCC cells radioresistance by activating PTEN/PI3K/AKT pathways.7. miR-20a induces cell resistance to irradiation treatment in vivo... |
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