Expression Of Hedgehog Signaling Components In Mouse Colorectal Cancer

Background：Colorectal cancer is a malignant tumor with high incidence. With the improvingliving standard and the changes in diets, the incidence of colorectal cancer in China ison the rise. Epidemiology data showed that China has become high risk areas forcolorectal cancer, which has400,000new cases every year，and patients between30-40years of age accounted for the majority. Therefore, investigating thepathogenesis and searching the key diagnostic and therapeutic targets of colorectalcancer have illustrated great theoretical and practical significance.Hedgehog signaling pathway makes great sense in organism development byregulating the embryonic development and the balance of cell proliferation andapoptosis. In addition, recent data showed that Hh signaling pathway play animportant role in tumorigenesis and development of many malignant tumors.Aberrant active Hh signaling pathway was found in colorectal cancer, but its exactmechanism in tumorigenesis and development of colorectal cancer still remainsunclear. Azoxymethane (AOM) is a chemical material which could specificallyinduce colorectal cancer. In this study, we establish colorectal cancer mouse modelwith AOM and dextran sulfate sodium (DSS), to investigate the regulatorymechanisms of Hh signaling in tumorigenesis and development of colorectal cancer,provide a new direction on developing targeted drug for colorectal cancer.Objective:1.Detect the expression level of SuFu, Gli1and Cyclin D1, three key moleculein Hh signaling pathway, in the mouse colorectal cancer model induced by AOM/DSSin order to assess the value of this mouse model in investigating the regulatatormechanism of Hh signaling in tumorigenesis and development of colorectal cancer.2. Construct a stable mouse colorectal cancer cell line expressing SuFu, which isthe negative factor of Hh signaling pathway, and to study effects of SuFu on Gli1、Cyclin D1and tumorigenesis of colorectal cancer. Methods:1. Mouse colorectal cancer model induced by AOM/DSSWeighed20-24g female BALB/c mice for this study, distributed into A/D group,AOM group, DSS group and Control group by random (28mice/group). On day1,inject intraperitoneally AOM working solution (12mg/kg body weight) for A/D andAOM group or sterile isotonic saline for the other two groups. From week2and on,mice of A/D and DSS group were watered with3%DSS (molecular weight5,000)solution, and distilled water for AOM and control group for1week every three weeksand repeated for3times. The mice were put to death at week24. The formation ofcolon neoplasm and pathological features certificated by HE strain are signs of tumorformation.2. General state of mouse(1) Weighed the mice every week for the first9weeks in the experiment,observed the stool to check for hematochezia or diarrhea.(2) Mice were put to death on week24, measured length of colon, observed theappearance of colon by naked eyes and pathological features by HE strain.3. Examined the expression of SuFu, Gli1and Cyclin D1in colorectal cancerand normal tissue with IHC.4. Constructed stable colorectal cancer cell line expressing SuFu(1) Constructed plasmid expressing SuFu named pUB6/V5-His-SuFu.(2) Transfected the pUB6/V5-His-SuFu plasmid to C26cell, and screened thestable cells expressing pUB6/V5-His-SuFu with Blasticidin. Verified the stable cellline by examing exogenous SuFu by Western Blot.5. Detected expression level of Gli1and Cyclin D1in C26-SuFu stable cell lineby Western Blot.Results:Animal Experiment1. After drinking3%DSS solution for the first time, there were23/27,24/28mice of A/D and DSS group suffered from hematochezia or diarrhea, respectively,and the ratio of the second time and the third time were22/27，21/26and17/20,18/23 respectively. The stools of those mice return to normal after restore normal drinkingwater. The stools of AOM and control group mice were always normal.2. The differences in weight changed among different mice groups had greatsignificance（F=16.523, p<0.01） after drinking3%DSS solution for the first time.The weight of A/D and DSS group decreased by1.33±1.52g and0.95±1.25grespectively, while AOM and control group increased by0.03±1.03g and0.95±0.92grespectively. But the weight of mice in each group had no difference during thesecond and third time of drinking3%DSS solution.3. At the end of week7,12mice of each group were sacrificed, but no lesionwas found. When putting the mice to death on week24, we could observe that thelength of colon of A/D and DSS mice were shorter than the control group. Neoplasmswere found in the colons of A/D group, but none in the other groups. HE strainshowed the pathological features of those neoplasms were accord with the perfor-mance of colorectal cancer.4. The difference of SuFu expression level among different groups wasstatistically significant (H=9.698, p<0.05). The SuFu expression levels of colorectalcancer tissue in A/D mice and normal colon tissue in AOM mice were highercompared to control group (p<0.05). The difference SuFu of expression levels had nostatistically significace (p>0.05) when compared pair wisely with other groups.5. The difference of Gli1expression level among different groups wasstatistically significant (H=9.663, p<0.05). The Gli1expression level of colorectalcancer tissue in A/D mice was higher compared to DSS and control group (p<0.05).The difference of Gli1expression levels had no statistically significace (p>0.05)when compared pair wisely with other groups.6. The difference of Cyclin D1expression level among different groups wasstatistically significant (H=10.039, p<0.05). The Cyclin D1expression level ofcolorectal cancer tissue in A/D mice was higher compared to DSS and control group(p<0.05). The difference of Cyclin D1expression levels had no statisticallysignificace (p>0.05) when compared pair wisely with other groups. Cell Experiment1. The PCR and DNA sequencing results showed that pUB6/V5-His-SuFuexpression plasmid was successfully constructed.2. The WB results showed that mouse colorectal stable cell line expressingSuFu (C26-SuFu) was successfully constructed.3. The expression levels of Gli1and Cyclin D1in C26-SuFu were lower thancontrol group (p <0.01).Conclusions:1. The mouse colorectal cancer model induced by AOM/DSS could be used toinvestigate the regulatory mechanisms of Hh signaling pathway in tumorigenesis anddevelopment of colorectal cancer.2. The mouse stable colorectal cell line expressing SuFu would be used to studythe role of SuFu in the tumorigenesis of colorectal cancer.3. Hedgehog signaling pathway was abnormally active in colorectal cancer.