| OBJECTIVE:The global incidence and mortality of liver cancer remain high.There are approximately 841,000 new cases and 782,000 deaths each year,of which 50% are from China,which seriously threatens human health.Searching for the potential pathogenesis and predisposing factors of liver cancer,early detection and early treatment is extremely urgent.The process of DEN(Diethylnitrosamine)inducing carcinogenesis is very similar to that of human liver cancer,and it is the most widely used liver cancer model.The occurrence and development of liver cancer is a complex multi-factor process involving abnormal changes at the cellular and molecular levels.Research on the pathogenesis of liver cancer mainly focuses on cell signal transduction,tumor-related gene expression regulation and so on.Gene chip can comprehensively analyze and judge the tissue specificity and disease specificity of gene expression,and quickly link genes and diseases.Although mi RNA is closely related to the occurrence of liver cancer,it has no obvious cell specificity.The piwi protein present in germ cells has tissue specificity.It has also been found in cancer cells in recent years.piRNA(Piwi-interacting RNA)that binds to piwi protein,It plays an important role in maintaining DNA integrity,inhibiting transcription,translation,etc.but the related research between piRNA/piwi and tumorigenesis is still in the initial stage.The main reason for the failure of chemotherapy for liver cancer is multi-drug resistance(MDR).Studies have shown that shikonin is a tumor suppressor,and it is not easy to develop drug resistance and has small side effects.But the molecular mechanism is not clear.In summary,this study took the rat liver cancer model induced by DEN as the research object,serum liver cancer markers,immunity,endocrine and related indicators were measured,and searched for differentially expressed mRNA,piRNA and related proteins in liver tissues,revealing the relationship between the above factors and occurrence and development of liver cancer;the liver cancer cell CBRH-7919 was used to detect the impact of shikonin on its proliferation,apoptosis,migration and invasion,hope it can provide theoretical support for exploring the molecular mechanism of liver cancer and clinical medication.Methods:1)Forty 6-week-old male healthy Wistar rats weighing about 150 grams were divided into liver cancer model group(NC)and normal control group(Md).Among them,the free drinking concentration of liver cancer model group was 0.1 mg/The sterile water of m L DEN should be updated every 24 hours,stop the drug after 20 weeks(replaced with water for 9-12 weeks).Observe the pathological changes of liver tissue and the changes of cell ultrastructure;detect peripheral Changes in blood AFP,ASMA,HSP,IL-1,IL-6,TNF-?,ACTH,CORT and other indicators by ELISA;using chip technology to detect the differential expression of liver tissue mRNA between groups and screen candidate genes,using RT-q PCR,immunization Histochemistry and Western-blot to conduct the detection of mRNA and protein expression levels of candidate genes in liver cancer tissues;2)Using next-generation sequencing to detect differentially expressed piRNAs in liver tissues;Using motif short sequence pattern matching algorithm to identify piRNAs adjacent to the regulatory region processing area Features are analyzed for enrichment and clustering,combined with piRNA location to summarize the distribution of functional genes,RT-q PCR was used to quantify piRNA and mi RNA,and detect piwil2,piwil4 and other genes and proteins expression level in liver tissue by immunohistochemistry and Western-blot,using CRISPR/cas9 gene editing system to knock out the piwil4 gene in CBRH-7919 cells,CCK-8,flow cytometry,and wound healing were used to detect changes in cell proliferation,apoptosis and migration;3)MTT,Flow cytometry,scratch experiment and transwell chamber method were used to observe the proliferation,apoptosis,migration and invasion after shikonin acts on CBRH-7919 cells,Western Blot was used to detect STAT3,Cyclin D1,piwil4,Bcl-xl protein after CBRH-7919 cells treated by shikonin.Results:1)DEN-induced liver cancer model in rats through improved drinking methods were successfully established.The carcinogenesis rate was 75%(15/20),and the death of experimental animals was effectively avoided.The liver surface roughness of the liver cancer group increased,false lobules,hyperplasia foci and a large number of cancers nodules formed.Compared with the normal control group,the serum AFP of the liver cancer model group was increased,and the difference was significant(P<0.01);there was no statistical difference in serum ASMA between the groups,and the serum HSP of the liver cancer model group and the normal control group were significantly different(P<0.01).Serum IL-1 and IL-6 levels decreased while TNF-? levels increased(P<0.05),and the levels of ACTH and CORT decreased.The mRNA microarray results showed that1639 of the 31042 mRNAs were up-regulated and 360 were down-regulated.The RT-q PCR method showed that the mRNA expression levels of STAT3 and Cyclin D1 genes were significantly increased(P<0.01);The mRNA expression levels of Igfals G6 PC were down-regulated,with statistical differences(P<0.05);immunohistochemical results showed that the expression of STAT3 and Cyclin D1 proteins in the normal control group was low,and the high expression of STAT3 and Cyclin D1 proteins in the liver cancer model group was mainly Localized in the nucleus.The western-blot experiment results showed that the expression levels of Igfals,G6 PC and Emp1 proteins were down-regulated,with statistical differences(P<0.05);2)A total of 27,439 piRNAs were found.The mRNA chip results showed that compared with the normal control group,there were 4 up-regulated piRNAs in the liver cancer model group(P<0.05),which were piR-64745(piR-rno-51044),piR-64265(piR-rno-50623),piR-79437(piR-rno-48177),piR-64744(piR-rno-51043),2 were down-regulated(P<0.01),which were piR-64143(piR-rno-50518)and piR-64142(piR-rno-50517).Abnormal expression of piRNA target genes between groups were involved in many pathways such as purine pyrimidine metabolism,amino acid degradation,mucopolysaccharide synthesis,replication,DNA shearing and repair,RNA and protein degradation,MAPK signaling pathway,and Ras signaling pathway.RT-q PCR showed that compared with the normal control group,the expression of piR-79437 in liver tissue increased(P<0.01),the expression of piR-64143decreased(P<0.01),and the serum mi R-133 b in the liver cancer model group was increased(P<0.05);The proteins of Piwil2 and Piwil4 were highly expressed(P<0.01).After Piwil4 gene knockout,the proliferation activity of cells in the KO group was reduced(P<0.05),and the apoptosis rate of the KO group was significantly increased(P<0.05),and the expression levels of STAT3,Cyclin D1,and Bcl-x L in Piwil4-/-cells were down-regulated,the difference was significant(P<0.05);3)Shikonin's IC50 was11.97?M,The concentration of the affected cells was determined to be 5,10,15?M,and the survival rate of the cells changed gradually with the increase of the drug concentration.the difference was significant(P < 0.05).The FCM results showed that when the concentration of shikonin was 0?M,5?M,10?M and 15?M,the number of apoptotic cells were 5.50±0.28%,7.88±0.51%,16.05±1.23%,26.88±0.22%,respectively,and the apoptotic rate gradually increased,showing a concentration-dependent different statistically(P<0.05),the wound healing results showed that the corresponding mobility of 0?M,5?M,10?M,15?M shikonin was 52.10±6.12%,47.01±9.01%,34.11±7.18%,13.98±4.52%,respectively.The concentration dependence was statistically significant(P<0.05);the transwell experiment showed that the number of cells passing through the membrane with 0?M,5?M,10?M,15?M shikonin was 482.33±55.54%,423.00±35.51%,386.33±68.37%,288.67±50.74% respectively.showing a concentration-dependent invasive ability decreased(P<0.05);after treating CBRH-7919 cells with 15?M shikonin for 48 hours,The expression of STAT3 and Cyclin D1 protein was down-regulated(P<0.05),and piwil4 and Bcl-x L did not change significantly from the normal control group.Conclusion:1)Using 0.1mg/ml DEN drinking water to induce rat liver cancer model for 20 weeks(withdrawal from 9-12 weeks),the carcinogenesis rate is 75%(15/20),and the effect is ideal.It was found that liver cancer occurred by pathological results of liver tissue,and serum AFP and HSP were both elevated.It is an ideal rat model for studying the occurrence and development of liver cancer.The author believes that the increased levels of immune-related IL-1,IL-6,and TNF-?,and the decrease of endocrine-related ACTH and CORT levels indicate that the immune and endocrine functions of liver cancer models are at least disturbed to some extent.2)In the model liver,the overexpression of STAT3,Cyclin D1 and other oncogenes and the low expression of the pro-apoptotic gene EMP1 together lead to the continuous proliferation of cells.The decrease of G6 PC expression in the liver cancer model facilitates the use of gluconeogenic intermediate products for the proliferation of liver cancer cells.It requires energy supply and material biosynthesis.3)Abnormal expression of piR-79437 and piR-64143 are associated with HCC and play a important role in the occurrence and development of liver cancer.Serum mi R-133 b may be a potential marker for liver cancer.The high expression of PIWIL2 and PIWIL4 in the liver cancer indicates that they have cancer-promoting effect.The expression levels of STAT3,cyclin D1 and Bcl-xl in PIWIL4 gene knockout cells are lower than those in the control group.STAT3/cyclin D1 promotes the proliferation of liver cancer cells and inhibits their apoptosis through STAT3/Bcl-x L.4)shikonin can promote the apoptosis,inhibit their migration and invasion of liver cancer cells,showing a concentration-dependent relationship,Shikonin down-regulates the expression of STAT3 and Cyclin D1 protein,indicating that it has an inhibitory effect on the growth of liver cancer cells,but there was no change in the expression levels of piwil4 and Bcl-x L after the cells treated by shikonin,suggesting that it does not affect cell apoptosis through Bcl-x L,and how to promote the apoptosis of liver cancer cells requires further research. |
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