| Liver cancer is one of the most common malignant tumors.In recent years,the incidence and mortality of liver cancer have been increasing year by year,and nearly half of the liver cancer patients in the world are in China,which has caused serious impacts on human health.With the increasingly mature clinical radiotherapy technology,stereotactic body radiotherapy(SBRT)has been widely used in the treatment of liver cancer.At present,the evaluation of the efficacy of liver cancer radiotherapy is mainly based on imaging data,or referring to the change level of traditional tumor markers,and lacks early and high-sensitivity prognostic markers.Many studies have confirmed that a variety of molecular markers are abnormally expressed in liver cancer,which is closely related to the occurrence and development of liver cancer,and has the potential to determine the biomarker of liver cancer radiotherapy prognosis.In this study,high-throughput sequencing analysis of transcriptomics changes in blood samples of clinical patients before and after SBRT,screening for new molecular markers and therapeutic targets,and targeted therapy verification through an orthotopic transplantation model of liver cancer to provide science for clinical radiotherapy in accordance with.ObjectiveTo find reliable molecular prognostic tumor markers for clinical SBRT treatment of primary liver cancer,and observe the therapeutic effect of targeted gene intervention combined with radiotherapy on orthotopic liver cancer transplantation in rats,to provides a scientific basis for evaluating the clinical efficacy of SBRT and finding new treatments for liver cancer.Methods1.Screening of tumor markers:17 primary liver cancer patients who met the inclusion criteria in a top three hospital were selected and clinical blood samples were collected(including 14 validated cases).The time point was before and after SBRT treatment for liver cancer patients 1(Before discharge after treatment)and 2 after treatment(2 months after treatment)blood samples were extracted RNA.Through high-throughput sequencing of transcriptomics(mRNA),molecules with significantly different expressions before and after SBRT treatment were selected.The screening criterion was q value(that is,corrected p value)<0.05 and 1.Collect patient medical records,imaging data 3 months after discharge,review the size of liver lesions,determine the outcome of liver cancer patients(CR,PR,SD or PD),classify CR and PR as SBRT effective group,SD and PD as SBRT invalid group.Combined with real-time quantitative qPCR detection of fluorescence,correlation analysis was performed to analyze the proportion of mRNA changes with significant differences,and observe whether the mRNA changes after SBRT were different between the effective group and the ineffective group.According to the curative effect after treatment and the results of qPCR detection before and after SBRT,the ROC curve was drawn,and the ability of different molecules as prognostic biomarkers for liver cancer SBRT was evaluated based on sensitivity and specificity.2.Experimental animals and grouping:Sixty-week-old healthy male Wistar strain rats were selected,weighing between 201-225g.The rat model of orthotopic liver cancer xenograft was constructed by surgery and randomly divided into sham irradiation group(Control),radiotherapy group(IR),empty virus control group(sh-NC),empty virus+radiotherapy group(sh-NC+IR),AdipoR1 silent group(sh-AdipoR1)and AdipoR1 silent+radiotherapy group(sh-AdipoR1+IR),10animals in each group.3.Establishment of animal model:(1)Preparation of liver cancer ascites:Resuscitate w256 cells,add 1.5mL of PBS buffer and mix well to make suspension.Prepare 4-5 week-old healthy male Wistar rats,draw 1 mL of cell suspension and inject into the abdominal cavity of the rat(the number of cells is about 1×10~6),and after about 6-7 days puncture to draw out the ascites,count the cells under the microscope,and prepare into rats In situ model of the required concentration(8×10~7/mL)of the suspension.(2)Tumor implantation:6-week-old healthy male Wistar rats were selected,weighing between 201-225g.Anesthetized rats were injected intraperitoneally with 5%chloral hydrate solution.The vertical incision in the upper left abdomen was about 1-1.5 cm to expose the liver.The left liver lobe was squeezed near the incision.Close the abdomen and suture the skin layer by layer.Fasting within12 hours after surgery,daily injection of 70,000 units of penicillin intraperitoneally for3 consecutive days.When the tumor diameter reaches about 15mm,the treatment is performed directly.4.Gene targeting and radiotherapy:(1)Virus solution configuration:After dissolving the LV-AdipoR1-RNAi virus solution and the negative control virus solution CON077 in ice bath,each virus is added to the HiTransG A virus infection reagent in a volume ratio of 1:3 Evenly.(2)Viral solution injection method:After the tumor is exposed,the virus solution is injected into the tumor body at multiple points,the abdomen is sutured layer by layer,and fasted within 12 hours after surgery.(3)adiotherapy:Place the anesthetized rat on the X-Rad 320 lifting plate,adjust the position of the rat so that the exposed tumor in situ is at the center of the lifting plate.The collimator has a diameter of 2 cm and the target center dose is 7 Gy.Set the irradiation plan:voltage 320kV,current 12.5mA,No.1 filter plate(aluminum),distance(SSD)50cm,dose rate 195.0cGy/min,single irradiation dose 7Gy,continuous irradiation for 3 days.After irradiation,the abdomen was closed and sutured.After fasting for 12 hours,the animals were fed with normal water.5.Evaluation indicators:observe and record the behavior and overall situation of rats after different treatment methods,measure the weight of the rats,measure the diameter of the tumor,judge the treatment effect,detect the genetic changes by real-time fluorescent quantitative PCR method,and calculate the spleen and thymus index Judging the immune function of rats,and measuring the blood routine and liver function indexes to judge the physiological status and the tumor killing effect of gene and radiotherapy rats.Results1.Screening of tumor markers for liver cancer:Analysis of changes in molecular expression before and after radiotherapy after full transcriptome sequencing revealed16 genes that remained elevated after SBRT treatment and 12 genes that remained decreased.The ratios of 28 mRNAs in the effective group and the ineffective group in two different stages were calculated,and the corresponding Z and P values were calculated.It was found that the two mRNAs of AdipoR1 and EPB42 were significantly different(p<0.05).All have potential as prognostic markers.According to the qPCR results before and after SBRT treatment,the ROC curve was drawn.The sensitivity of the theoretical values of the AdipoR1 and EPB42 genes were 100%and75%,the Yoden index was 0.83 and 0.75,and the actual sensitivity was 60%and 80%,respectively.Suggesting that the two novel markers AdipoR1 and EPB42 genes can be used to evaluate the prognosis of liver cancer SBRT.2.The overall performance of the rats after treatment:There was no ascites and metastasis of cancer cells in the rats in each group.The food and drinking conditions of the rats in each group were normal,and no symptoms of intestinal radiation sickness such as diarrhea were seen.The control group,the sh-NC group and the sh-AdipoR1 group rats had significant differences compared with the three groups of rats after radiation treatment 7 days after the treatment:the body weight of the rats after radiation treatment did not change significantly,The mental state is good,the hair gloss has not changed,and the weight of the three groups of Control,sh-NC and sh-AdipoR1 rats has decreased to a certain extent,drinking water,less activity,and some of them have vertical hair performance.Seven days after the end of the treatment,the sh-NC group showed a certain degree of weight loss compared with the sh-AdipoR1 group;the IR group also showed weight loss compared with the sh-AdipoR1+IR group.To a certain extent(p<0.05).3.Inhibition of tumor growth by various treatment factors:Real-time quantitative PCR confirmed the silencing effect of AdipoR1 gene.The expression of AdipoR1gene in the IR group and sh-NC+IR group increased compared with the Control group,sh-AdipoR1 The gene expression of AdipoR1 in the group and sh-AdipoR1+IR group was significantly lower than that in the Control group(p<0.05).After 7days of radiotherapy,the tumor volume of rats had a significant decrease compared with no radiotherapy(Control group,sh-NC group,sh-AdipoR1 group),suggesting that radiotherapy effectively inhibited tumor growth and development;Compared with the sh-NC group,the sh-AdipoR1 group's tumor volume also decreased after treatment(p<0.05).The sh-AdipoR1+IR group's tumor volume was also smaller than that of the IR group.Except for the Control group and the sh-NC group,the tumor volume of rats in other groups all decreased to varying degrees after treatment,and the tumor diameter reduction was most obvious in the sh-AdipoR1+IR treatment.4.The effects of various treatment factors on the spleen and thymus index:IR group and Control group,sh-NC+IR group and sh-NC group,sh-AdipoR1+IR group and sh-AdipoR1 group can be found by comparing,The spleen index and thymus index of rats were significantly increased(p<0.05).The spleen and thymus index of the rats in the sh-NC group were not statistically different from those of the sh-AdipoR1 group.The thymus index did not change significantly compared with the sh-AdipoR1+IR group.5.The influence of various treatment factors on blood routine indicators:Compared with the Control group,the red blood cell number,hemoglobin content and platelet number in the blood of the IR group were significantly increased(p<0.05);Compared with the sh-NC group,the number of white blood cells in the sh-NC+IR group decreased significantly,and the other three indicators increased significantly;when AdipoR1 was silenced,the number of red blood cells,hemoglobin content,and white blood cells were not statistically different.The number increase is reflected(p<0.05),which is the only group of rats with an increased white blood cell count;when AdipoR1 is silenced and combined with radiotherapy,except for the red blood cell count that does not show statistical differences and the white blood cell count decreases,the hemoglobin content And platelet counts showed a tendency to improve.After radiation therapy,the number of red blood cells and hemoglobin increased,and the anemia was relieved.Silencing the AdipoR1 gene can also significantly reduce the symptoms of anemia in rats,while increasing the number of white blood cells in the body to improve immune resistance.The combination of the two treatment methods has the highest hemoglobin content and a significant effect;at the same time,increased platelet production also benefits from IR or sh-AdipoR1+IR.6.The influence of various treatment factors on liver function indicators:except that the sh-NC+IR group did not show statistical differences,the ALT of rats in the IR group,sh-AdipoR1 group,and sh-AdipoR1+IR group all had The decline was the most in the sh-AdipoR1+IR group.Although in AdipoR1 silenced rats,the addition of radiotherapy did not show a statistical difference,but other treatment methods can significantly reduce the content of AST in serum.Although the sh-AdipoR1 silence group has a lower ALP value than the sh-NC group,it does not show a statistical difference,but the IR group and the sh-AdipoR1+IR group can prove that radiotherapy or silence AdipoR1 gene therapy is added to a certain extent.ALP improves liver function.The albumin in the sh-AdipoR1+IR group reached the highest level,which was equivalent to the sh-AdipoR1 group.Conclusions1.This study screened two new tumor prognostic markers(AdipoR1 and EPB42)for liver cancer,which are highly sensitive and specific biomarkers that can be used to evaluate the prognosis of SBRT in liver cancer patients.2.Silencing the expression of AdipoR1 gene can improve the survival status of liver cancer model rats and has a significant inhibitory effect on tumors.3.Compared with radiotherapy alone,silencing AdipoR1 gene-targeted combined radiotherapy has a better local control rate for rat liver cancer,can significantly improve the prognosis of rats,and has more significant therapeutic effects.Further research on AdipoR1 may bring new opportunities for clinical diagnosis and treatment of primary liver cancer. |
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