| Hepatocellular carcinoma（HCC）dominated primary hepatocellular carcinoma ranks the sixth in the incidence of malignancies and the fourth in mortality in the world.Radiotherapy,as an important local treatment for HCC,has been widely used in clinical practice.In clinical practice,due to the large size of the lesions in most HCC patients and the fact that the HCC is often accompanied by hypoxic necrosis,the radiotherapy sensitivity is significantly reduced.Most of the patients in China are accompanied by chronic viral hepatitis,cirrhosis and other basic liver diseases,resulting in poor tolerance of normal liver cell tissues to radiation.The spinal cord,stomach and small intestine around the liver cancer lesions are all radiationsensitive organs,and it is difficult to give the radical radiotherapy dose to the lesions clinically,which ultimately leads to the unsatisfactory radiotherapy effect.Therefore,the study on the regulation of energy metabolism and radiation resistance mechanism of HCC under hypoxic conditions is conducive to improving the efficacy of local radiotherapy for HCC and providing a new molecular target for targeted therapy.Research background:Ionizing radiation kills tumor cells by indirectly inducing oxygen free radicals to induce double and single strand DNA damage in tumor cells.Hypoxia in tumor microenvironment leads to a significant reduction of reactive oxygen species and oxygen free radicals in tumor cells,leading to the occurrence of radiation resistance.Even in the case of sufficient oxygen supply and normal mitochondrial function,tumor cells still use aerobic glycolysis to absorb glucose and convert it into lactic acid,instead of using oxidative phosphorylation for energy supply.This phenomenon is called the warburg effect,and it is the main feature of energy metabolism of malignant tumor cells.The accumulation of acidic substances such as lactate,pyruvate,glutathione and NADPH in aerobic glycolysis,these oxidizing reductants can effectively remove reactive oxygen species and oxygen free radicals,up-regulate the endogenous antioxidant capacity,and further lead to the occurrence of radiation resistance.Micro RNA is an important regulatory factor for tumor genesis and development.Many scholars have found that mi R-873 is abnormally expressed in different malignancies such as glioblastoma,colorectal cancer and lung adenocarcinoma,and plays the role of its oncogene or tumor suppressor gene by targeting and regulating different downstream genes.It has been reported that mi R-873 can participate in the regulation of NF-κB、ERK、PI3K/AKT、Wnt/β-catenin and other signaling pathways.However,the expression of mi R-873 in the development of HCC and its biological functions in the relationship between energy metabolism and radiation resistance of HCC have not been clearly reported.Research objectives:Tumor cells maintain their rapid and continuous proliferation through the warburg effect and promote the occurrence of radiation resistance.Elucidating the regulation of energy metabolism and the occurrence of radiation resistance of HCC in hypoxic microenvironment,as well as the relevant molecular regulation mechanism,is of great significance for improving the efficacy of local radiotherapy for HCC.Research methods:Based on the above research status,this study firstly detected the expression of mi R-873 in 86 samples of HCC tissues（tumor tissues and paracancer tissues）,and conducted statistical analysis on the correlation with clinical information.Cell proliferation,migration and invasion experiments were used to investigate the biological function of mi R-873 in HCC.The effects of mi R-873 on the radiation resistance of HCC were investigated by cell cloning,MTT and flow cytometry.Mi R-873 energy metabolism pathway and related indexes were analyzed and its relationship with the wahlberg effect was elucidated by means of glucose metabolism correlation experiment and gas chromatography-mass spectrometry（GC-MS）analysis to trace the metabolite flux of 13C-labeled glucose.The public mi RNA database was used to predict that the target gene for its downstream regulation was NDFIP1,and the targeting relationship between the two was verified by means of luciferase reporter gene,q RT-PCR and immunohistochemistry.The above experiments were repeated by gain-and-loss method to confirm the relationship between the biological function of NDFIP1 and the warburg effect in the occurrence and development of HCC.Finally,Western Blot,q RT-PCR,IHC and other methods were used to detect the up-stream and down-stream regulatory signaling molecules in vivo and in vitro.Research results:1.The expression level of mi R-873 in HCC and its correlation with clinical information were detected in vivo and in vitro（1）The correlation between the expression of mi R-873 in HCC tissues and the clinical informationThis study collected 86 patients with HCC tumor and cancer adjacent tissues and to detect the expression of mi R-873,and in-depth analysis of the expression levels of mi R-873 in HCC patients with basic information such as information,tumor related indicators and clinical prognosis of patients（age,gender,tumor size and pathology classification,clinical stage and lymphatic vascular metastasis and survival and recurrence time clinical prognosis）,statistical analysis of the clinical significance of mi R-873 expression in HCC.The results showed that mi R-873 was highly expressed in liver cancer tissues,which was significantly higher than that in paracancer tissues.Moreover,the expression of mi R-873 was positively correlated with the malignant degree of tumor,such as clinical stage,lymph node metastasis and distant metastasis,and negatively correlated with the degree of tumor differentiation,and negatively correlated with the survival and recurrence time of HCC patients.Multivariate analysis showed that the clinical stage of HCC and the expression level of mi R-873 were adverse prognostic factors.（2）The expression of mi R-873 in HCC cells and normal cellsq RT-PCR was used to detect the expression levels of mi R-873 in 5 liver cancer cell lines（SMMC-7721,Hep G2,Hep3 B,SK-HEP-1,MHCC97H）and 3 human normal liver cell lines（L02,7701,7702）,and it was found that compared with normal liver cell lines,mi R-873 in liver cancer cell lines was highly expressed（p<0.01）.In conclusion,mi R-873 may play an important role as an oncogene in HCC.2.To investigate the biological function of mi R-873 in HCC（1）The effect of overexpression of mi R-873 gene on the proliferation,migration and invasion of HCCmi R-873 mimic were transfected into SMMC-7721 cells by retrovirus transfection to construct a stable mi R-873 overexpression SMMC-7721 cell line（SMMC-7721-mi R-873high）.After successful transfection was verified by q RT-PCR,its proliferation,migration and invasion were detected by CCK-8 and Transwell experiments.Results analysis showed that compared with SMMC-7721-NC cells,SMMC-7721-mi R-873 high cells had stronger proliferation,migration and invasion ability（p<0.01）.（2）Inhibition of mi R-873 gene on the proliferation,migration and invasion of HCCmi R-873 inhibitor#1 and #2 were transfected into Hep3 B cells by retrovirus transfection to construct stable Hep3 B cell lines with low expression of mi R-873（Hep3B-anti-mi R-873-# 1 and Hep3B-anti-mi R-873-#2）.After successful transfection was verified by q RT-PCR,the proliferation,migration and invasion abilities of Hep3 B cells and normal Hep3 B cells with low expression of mi R-873 were also detected by CCK-8 and Transwell experiments.The results showed that,compared with Hep3B-NC cells,Hep3 B cells with low expression of mi R-873 significantly inhibited cell proliferation,migration and invasion（p<0.01）.Combined with the above experimental results,it can be concluded that mi R-873,as an oncogene in HCC,has biological functions to promote the proliferation,migration and invasion of HCC.3.To investigate the effect of mi R-873 on radiation resistance of HCC（1）Effects of overexpression of mi R-873 on radiation resistance of HCCmi R-873 mimic was transfected into SMMC-7721 cells to overexpress mi R-873,and the proliferation ability of SMMC-7721 cells was detected by MTT assay at 24 and 48 h after 10-Gy X-ray irradiation.Compared with the transfected NC control group,the survival rate of SMMC-7721 cells that overexpressed mi R-873 was increased by about 15%（p<0.05）.The results of cell cloning formation experiments after 0,1,2,4,6 and 8 Gy X-ray irradiation showed that the survival scores of SMMC-7721 cells that overexpressed mi R-873 significantly increased.Flow cytometry was used to detect SMMC-7721 cells that overexpressed mi R-873 after 6 Gy X-ray irradiation,and the results showed that the apoptosis rate significantly decreased（p<0.01）.It was shown that overexpression of mi R-873 could produce radiation resistance.（2）The effect of inhibiting the expression of mi R-873 on the radiation resistance of HCCAfter Hep3 B cells were transfected with anti-mi R-873 to inhibit the expression of mi R-873,the proliferation ability of cells was detected by MTT method at 24 and 48 h after 10 Gy X-ray irradiation.The results showed that the survival rate of Hep3 B cells with low mi R-873 expression decreased by about 15%（p<0.05）compared with those transfected with antiNC.After X-ray irradiation of 0,1,2,4,6 and 8 Gy,cell cloning and formation experiments showed that the survival scores of Hep3 B cells inhibiting mi R-873 were significantly reduced.Flow cytometry was used to detect the apoptosis of Hep3 B cells that inhibited mi R-873 after 6 Gy X-ray irradiation.The results showed that the apoptosis rate was significantly increased and G2/M phase arrest occurred in the cell cycle（p<0.05）.It was suggested that inhibition of mi R-873 expression could reduce the radiation resistance of HCC.In summary,the experimental results showed that overexpression of mi R-873 in HCC resulted in radiation resistance,while inhibition of mi R-873 significantly reduced the radiation resistance of HCC.4.To investigate the regulatory function of mi R-873 on glucose metabolism in HCC（1）Effects of overexpression or inhibition of mi R-873 on glucose metabolism of HCCGlucose metabolism indexes such as extracellular acidification rate（ECAR）,oxygen consumption rate（OCR）,glucose intake and lactic acid production were analyzed.The results showed that both the basal ECAR and the maximum ECAR of Hep3 B cells that inhibited the expression of mi R-873 gene were significantly reduced,while OCR was significantly increased（p<0.01）,and both glucose uptake and lactic acid production showed a decreasing trend（p<0.05）.Similarly,mimic overexpression of mi R-873 was transfected into SMMC-7721 cells and the above-mentioned glucose metabolism indexes were detected.The results showed that ECAR,glucose uptake and lactic acid production all showed an increasing trend after overexpression of mi R-873（p<0.01）,while OCR decreased（p<0.01）.In conclusion,mi R-873 enhanced the glucose metabolism of HCC.（2）The involvement of mi R-873 in the regulation of aerobic glycolysis of HCC was verified13C-labeled glucose metabolic fluxes in SMMC-7721 cells that overexpressed mi R-873 were tracked by gas chromatography-mass spectrometry（GC-MS）.The results showed that the 13C-labeled glucose level in SMMC-7721 cell medium with overexpression of mi R-873 significantly decreased,and the 13C-labeled intracellular and extracellular lactic acid levels increased（p<0.05）,and the overexpression of mi R-873 promoted the aerobic glycolysis of HCC.Detected at the same time,the intermediate metabolites levels of glycolysis such as the 13C-labeled glucose-6-phosphate（G6P）,1,6 diphosphate（F1,6BP）and phosphoenolpyruvate（PEP）rise,and intermediate metabolites levels in tricarboxylic acid cycle（TCA）such as citriate,succinate,fumarate,and malate decline（p<0.05）,in conclusion the results showed that mi R-873 in HCC promote aerobic glycolysis,rather than the oxidative phosphorylation.5.Regulatory target genes and biological functions of mi R-873（1）The target gene of mi R-873 were predicted and verified1)Bioinformatics predicts the target gene regulated by mi R-873The public mi RNA database（Target Scan,mi Randa,Target Rank）was used to predict that the target gene of mi R-873 was NDFIP1 through the 3’-UTRs gene locus associated with glycolysis.By cloning the 3’-UTR of NDFIP1 containing the mi R-873 target site into the luciferase reporter vector,the luciferase reporter gene was used to verify the targeting relationship between the two.2)Confirmed the regulatory effect of mi R-873 on NDFIP1After transient transfection of HEK293 T cells with different concentrations of pre-mi R-873,the levels of endogenous NDFIP1 protein were detected by Western Blot.The results showed that the transfection amount of mi R-873 was negatively correlated with NDFIP1（p <0.01）.q RT-PCR and immunohistochemical staining were used to detect the expressions of mi R-873 and NDFIP1 in 20 pairs of clinical HCC samples.It was found that the m RNA level of NDFIP1 in HCC was significantly lower than that in para-carcinoma tissues,and the expression of mi R-873 in HCC was found to be negatively correlated with NDFIP1（p =0.008）.（2）To investigate the effect of NDFIP1 as a target gene of mi R-873 on the glycolysis of HCCAfter overexpression of mi R-873 and/or NDFIP1 in SMMC-7721 cells,the above glucose metabolism correlation was detected,and the experiment was divided into four groups（NC,mi R-873,mi R-873+vector,mi R-873+NDFIP1）.The results showed that through the detection of ECAR,OCR,glucose intake,lactate production and other indicators,it was found that overexpression of mi R-873 could enhance aerobic glycolysis,while overexpression of NDFIP1 could significantly reduce the glycolysis enhanced by overexpression of mi R-873（p <0.01）.The results showed that mi R-873 promoted aerobic glycolysis by inhibiting NDFIP1.（3）To investigate the biological function of NDFIP1 as a target gene of mi R-873 in HCCThe biological functions of NDFIP1 in the proliferation,migration and invasion of HCC were explored through the gain-and-loss experiment.p CDH-CMV-NDFIP1 was overexpressed in SMMC-7721 cells.The results of cell proliferation experiment and Transwell experiment showed that compared with SMMC-7721-NC cells,SMMC-7721 cells with overexpression of NDFIP1 significantly reduced the ability of proliferation,migration and invasion（p<0.01）.Both NDFIP1 and mi R-873 were overexpressed in SMMC-7721 cells.The results of cell proliferation assay and Transwell assay showed that SMMC-7721 cells with overexpression of these two genes significantly reduced the ability of proliferation,invasion and migration compared with SMMC-7721 cells with overexpression of mi R-873 alone（p <0.01）.si RNA was used to inhibit NDFIP1 and anti-mi R-873 in Hep3 B cells to inhibit the expression of mi R-873,and the results of cell proliferation experiment and Transwell experiment showed that Hep3 B cells that inhibited the expression of two genes at the same time could significantly enhance the proliferation,migration and transfer of cells compared with Hep3 B cells that inhibited mi R-873 alone（p<0.01）.These results suggest that mi R-873 plays the biological role of an oncogene that promotes HCC proliferation,migration and invasion by inhibiting the expression of NDFIP1.6.Elucidate the signaling mechanism of mi R-873 / NDFIP1 regulating the warburg effectWestern Blot was used to detect the expression of HIF-1α,c-Myc,AKT-s473,S6 K and other key proteins that regulate the glucose metabolism signaling pathway.The results showed that compared with the control group,there was no change in the levels of HIF-1α,c-Myc and total AKT in SMMC-7721 cells that overexpressed mi R-873,but the phosphorylation of AKTs473 and S6 K was induced.The overexpression of mi R-873 in SMMC-7721 cells was treated with a PI3 K specific inhibitor（LY294002）,and the expression of related proteins was detected by Western Blot.The results showed that LY294002 significantly inhibited the phosphorylation of AKT and decreased the expression of the key enzymes Glut1 and HK2 in the mi R-873-induced enhanced glycolysis pathway（p<0.01）.Both m TOR inhibitors（rapamycin）and LY294002 inhibited the increase of glucose uptake and lactic acid production in SMMC-7721 cells with mi R-873 overexpression（p<0.05）.In summary,the above data indicate that the activation of AKT or m TOR is necessary for the induction of aerobic glycolysis by mi R-873,indicating that its downstream regulatory signaling pathway is the AKT/m TOR signaling pathway.7.Elucidate the upstream regulatory molecular mechanism of mi R-873 in HCCThe expression of mi R-873 in HCC under Co Cl2 simulated hypoxia was detected by q RT-PCR.The results showed that the m RNA expression levels of mi R-873 in SMMC-7721 and Hep3 B cells were significantly up-regulated after Co Cl2 treatment（p<0.01）.In the above two kinds of cells,the expression of HIF-1α was decreased by transfection of the two kinds of si-HIF-1α expression.After the same Co Cl2 treatment,the expression of mi R-873 m RNA was detected by q RT-PCR,and the expression of mi R-873 was significantly down-regulated（p<0.05）.To further clarify how HIF-1α regulates the expression of mi R-873,we used mi RStart and JASPAR databases to predict the TSS and HRE of mi R-873,and found that HIF-1α binds to HREs in the-1kb sequence region upstream of TSS in mi R-873.Therefore,the mi R-873（-1 KB）promoter region containing the predicted HRE sequence was cloned into the p GL3 vector.Similarly,the luciferase activity of mi R-873 promoter increased after the treatment of HEK293 T cells with COCl2,while the luciferase activity was significantly decreased after the transfection of si-HIF-1α（p<0.01）.The effect of HIF-1α on the regulation of mi R-873 was further confirmed by subcutaneous tumor transplantation in nude mice.Mice of BALB/c nude mice of Hep3 B subcutaneous xenograft tumor were sacrificed after continuous gavage for 15 days using HIF-1α specific inhibitor（BAY87-2243）.The m RNA expression level of mi R-873 in tumor tissues was detected by q RT-PCR and the expression of HIF-1α protein was detected by IHC.The results showed that the m RNA expression of mi R-873 in BAY87-2243 treated mice was significantly reduced（p<0.01）,and the expression of HIF-1α protein was significantly decreased in IHC compared with the control mice.Conclusion:1.mi R-873 is highly expressed in HCC,which is positively correlated with the degree of HCC malignancy and negatively correlated with clinical prognosis.2.As an oncogene,mi R-873 plays a biological role in promoting the proliferation,migration and invasion of HCC;3.Overexpression of mi R-873 produces radiation resistance,and targeted inhibition of mi R-873 can significantly reduce the radiation resistance of HCC,which is conducive to improving the radiosensitivity of HCC.4.mi R-873 enhances the Warburg effect by enhancing the aerobic glycolysis of HCC and inhibiting the oxidative phosphorylation;5.NDFIP1 is a target gene of mi R-873,which acts as a tumor suppressant gene and plays a biological role in inhibiting the proliferation,migration,invasion and Warburg effect of HCC.6.mi R-873 /NDFIP1 axis enhances the Warberg effect of HCC through the AKT/m TOR signaling pathway,thereby promoting the growth and metastasis of HCC;7.In the hypoxic HCC microenvironment,overexpression of HIF-1α is induced and the expression of mi R-873 is upregulated,thereby regulating the occurrence of Warburg effect and promoting the proliferation,migration,invasion and radiation resistance of HCC. |
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