| Objective:To explore the inhibitory effect of activating blood circulation to dissipate blood stasis recipe on colorectal cancer and the regulation mechanism of zDHHC9/RAS through theoretical research,cell experiment and clinical research,so as to provide objective basis for TCM syndrome differentiation in the treatment of colorectal cancer.Methods:1.Theoretical research:to explore the mechanism of RAS signal transductionmechanisms,the current situation of western medicine related research,and TCM syndrome differentiation and treatment of colorectal cancer through consulting the relevant literature.2.Experiment research:1)to compare the expression of zDHHC9 in normal tissue cells and colon cancer cell lines;2)to detect the effect of zDHHC9 on proliferation of colon cancer cells and Ras signaling pathway by silencing and overexpression of zDHHC9;3)Three doses of activating blood circulation to dissipate blood stasis recipe were selected to intervene SW620 colon cancer cell line to observe the efects of activating blood circulation to dissipate blood stasis recipe on growth,cell cycle,apoptosis,expression of zDHHC9 and Ras signaling pathway of SW620 colon cancer cell line.3.Clinical research:1)the expression of zDHHC9 and RAS mutation in colorectal cancer tissues and serum were retrospectively analyzed,and the relationship between the expression of zDHHC9 and clinical pathological signs,MSI status,survival time and treatment sensitivity was statistically analyzed.2)activating blood circulation to dissipate blood stasis recipe combined with western medicine was used to treat colorectal cancer patients with stagnation of qi and blood stasis to observe the objective effective rate,zDHHC9 change,TCM syndrome score.Results:1.Theoretical research:1)after consulting a large number of literatures,it is found that RAS related inhibitors are still in progress,and there is no effective method for RAS mutation.2)zDHHC9 may be the acyltransferase protein of human RAS protein,and it is most overexpressed in colorectal cancer.RAS may be catalyzed and regulated by palmitoyl acyltransferase protein zDHHC9 in colorectal cancer.To further understand whether zDHHC9 acts as the palmitoyl acyltransferase of RAS in colorectal cancer to regulate RAS to play its oncogene effect is particularly important for clarifying the pathogenesis of colorectal cancer,guiding individualized treatment and researching new therapeutic targets.3)In the treatment of colorectal cancer,traditional Chinese medicine mostly adopts the method of strengthen body resistance and eliminating evil,strengthening spleen and tonifying kidney is the most common method for strengthening the body,and activating blood circulation to dissipate blood stasis is the most common method for removing pathogenic factors.Based on previous studies,it was found that Huoxuehuayu Recipe has a strong inhibitory effect on drug-resistant colorectal cancer cell lines with high expression of zDHHC9,and further research is needed to clarify the mechanism of action.2.Experiment research:1)the expression of zDHHC9 protein in colon cancer cell lines was in different degrees,but it was almost not expressed in normal tissue cells.2)CCK8 test and 3D Matrigel test showed that the expression level of zDHHC9 was positively correlated with cell proliferation.The results of Western blot showed that zDHHC9 knockdown could reduce the phosphorylation of ERK in RAS downstream pathway.Confocal microscopy showed that zDHHC9 was involved in regulating the expression of Ras in cell membrane,and the expression of Ras on the surface of cell membrane with high expression of zDHHC9 was increased.3)Activating blood circulation to dissipate blood stasis recipe inhibited the growth of SW620 cells and the expression of zDHHC9,but had no significant effect on cell cycle and apoptosis.3.Clinical research:1)the positive rate of zDHHC9 in colorectal cancer was significantly higher than that in colorectal polyps(P=0.001)and normal control group(P<0.001).In different stages,compared with stage ?,? and ?,the expression of zDHHC9 in stage ? colorectal cancer was the highest(P<0.01).The zDHHC9 positive expression rate of RAS mutation patients and all wild-type patients was higher.The positive expression rate of BRAF mutation patients was lower(P=0.03).There was no significant difference between male and female gender and age.The expression of zDHHC9 was different among different microsatellite states.The positive expression rate of zDHHC9 was higher in MSS colorectal cancer than in MSI-H colorectal cancer(P<0.01).The survival rate of zDHHC9 positive patients was lower,and the survival rate of negative patients was higher,but the difference was not statistically significant(P>0.05).The overall effective rate of zDHHC9 positive patients was higher,but the difference was not statistically significant(P>0.05).2)Activating blood circulation to dissipate blood stasis recipe combined with western medicine in the treatment of colorectal cancer patients with stagnation of qi and blood stasis:there was no significant difference in objective response rate and disease control rate between the western medicine treatment group and the traditional Chinese and Western medicine treatment group(P>0.05).Traditional Chinese and Western medicine group improved the TCM syndrome score significantly,and the difference was statistically significant(P=0.02).The reduction of zDHHC9 expression in the traditional Chinese and Western medicine group was greater than that in the western medicine group,the difference was statistically significant(P=0.04).Among the patients in the traditional Chinese and Western medicine group,the objective effective rate of zDHHC9 positive patients was 48.6%;the objective effective rate of zDHHC9 negative patients was 23.1%.Patients with zDHHC9 positive are more effective.The difference was statistically significant(P=0.04).In the cases of traditional Chinese and western medicine group,the expression of serum zDHHC9 in patients with KRAS/NRAS mutation and all wild-type patients before treatment was similar,and both were higher than those of BRAF mutantpatients(P<0.05).zDHHC9 expression decreasedin patients with KRAS/NRAS mutation and all wild-type patientsafter treatment,and the difference was statistically significant(P=0.04).zDHHC9 expression in BRAF mutant patients was the lowest,and zDHHC9 did not change significantly after treatment(P>0.05).Conclusion:1.zDHHC9 is highly expressed in colorectal cancer cell lines,which can regulate the location of RAS in the cell membrane and the expression of downstream signal ERK,and control cell proliferation.2.Huoxuehuayu Recipe can inhibit the growth of colon cancer cells,which may be related to inhibiting the expression of zDHHC9 and reducing the phosphorylation level of ERK downstream of RAS.3.The positive rate of zDHHC9 expression in colorectal cancer tissue was significantly higher than that in colorectal polyp group and normal control group.Stage IV colorectal cancer had the highest expression,and the positive expression rate of MSS colorectal cancer was higher than that of MSI-H.4.Compared with Western medicine alone,Huoxuehuayu Decoction combined with Western medicine can significantly improve TCM-related syndromes.Patients with high expression of zDHHC9 have better therapeutic effects,and patients with effective treatment have significantly lower zDHHC9.KRAS/NRAS mutant population has a high effective rate,which may be related to the high expression of zDHHC9 regulating the RAS signaling pathway. |
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