The Role Of AQP8 In Colorectal Cancer And Its Effect On PI3K/Akt Signal And PCDH7 Expression

Background:Canceration and metastasis of colorectal cells are multistage processes.Aquaporin?AQPs?consists of a family of membrane transporters,which play an important role in water transport.AQP8 is a kind of aquaporin,which not only transports water,but also promotes ammonia and hydrogen peroxide to pass through cell membranes.In addition to the function of membrane channel,AQP8 also has a variety of biological functions,thus affecting the body's physiological functions and disease progression.In addition,AQP8 is closely related to the occurrence and metastasis of ovarian cancer and breast cancer.Other studies have shown that the expression of AQP8 in human colorectal cancer is decreased,suggesting that the AQP8 gene is down-regulated during tumorigenesis.PI3K-Akt pathway is one of the most common disordered signaling pathways in all cancers.Some AQPs also affect PI3K/Akt signal-related proteins in the process of affecting cancer and other diseases.Human PCDH7 is a member of the cadherin superfamily and plays an important role in cell recognition,adhesion and signal transduction.A large number of studies have shown that PCDH7 expression is disordered or mutated in various types of cancer tissues or cells.Some studies also showed that there was a co-expression relationship between PCDH7 and AQP8.In this paper,we studied the significance of AQP8 in the occurrence and development of colorectal cancer through population,animal and cell experiments,and explored the relationship between AQP8 and PI3K/Akt signal and PCDH7.Part ? Expression and clinical significance of AQP8,PCDH7 and PI3K/Akt signal-related proteins in colorectal cancerAIM:To study the expression of AQP8,PCDH7 and Akt in colorectal cancer and adjacent tissues,and to explore the relationship between the three indicators and clinical characteristics and prognosis of patients.Methods:From June 2016 to December 2016,56 patients with colorectal cancer were collected in Guangdong General Hospital.In the HIS case inquiry system,the age,tumor size and lymph node metastasis of patients at the time of initial diagnosis were inquired.We used self-matching method to extract colorectal cancer tissues and adjacent normal tissues for each patient(distance from the tumor?>5 cm?.From the time of diagnosis,all the subjects were followed up regularly with an interval of 4 weeks.Western blot was used to detect the expression of AQP8,PCDH7 and Akt in tissues.RT-PCR was used to detect the expression of AQP8,PCDH7 and Akt in tissues.Result:1.The relative expression level of AQP8 in cancer tissue was significantly lower than that in adjacent tissues.The relative expression levels of PCDH7 and Akt in cancer tissue were significantly higher than that in adjacent tissues.2.The relative expression levels of AQP8 protein in cancer tissues were significantly lower than those in adjacent tissues.The relative expression levels of PCDH7 and Akt protein in cancer tissues were significantly higher than those in adjacent tissues.3.AQP8 protein was negatively correlated with PCDH7 protein?correlation coefficient=-0.473,P=0.000?;AQP8 protein was negatively correlated with Akt protein?correlation coefficient=-0.676,P=0.000?;PCDH7 protein was positively correlated with Akt protein?correlation coefficient=0.544,P=0.000?.4.The relative expression level of AQP8 protein in colorectal cancer tissues with malignant intestinal obstruction,lymph node metastasis,tumor diameter greater than 5 cm,C+D stage and highly differentiated was significantly decreased?P<0.05?.The relative expression level of PCDH7 protein in colorectal cancer tissues with carcinomatous intestinal obstruction,lymph node metastasis,tumor diameter greater than 5 cm and C+D stage was significantly higher?P<0.05?,but had no significant correlation with the degree of cancer differentiation?P>0.05?.The relative expression levels of Akt protein in colorectal cancer tissues with intestinal obstruction,lymph node metastasis,tumor diameter greater than 5 cm,C+D stage and highly differentiated were significantly increased?P<0.05?.5.AQP8 protein level was significantly decreased in cancer tissues of relapsed patients,while PCDH7 and Akt protein were significantly increased.6.The survival time of patients with high expression of AQP8 was significantly higher than that of patients with low expression of AQP8;the survival time of patients with high expression of PCDH7 was significantly lower than that of patients with low expression of PCDH7;and the survival time of patients with high expression of Akt was significantly lower than that of patients with low expression of Akt.CONCLUSION:AQP8 is low in colorectal cancer tissues and PCDH7 and Akt are high in colorectal cancer tissues.The three indices were correlated with the clinical characteristics,recurrence and prognosis of the patients.High expression of AQP8 protein is a protective factor for the prognosis of patients with colorectal cancer.High expression of PCDH7 and Akt protein is a prognostic risk factor.AQP8 protein was negatively correlated with PCDH7 protein and Akt protein.Part ?:The effect of AQP8 on the biological function of rectal cancer cells and its regulation on PCDH7 and PI3K/Akt signalsAIM:To study the effects of AQP8 on proliferation,apoptosis,invasion and migration of colorectal cancer cells in cell experiments.The regulatory effects of AQP8 on PCDH7 and PI3K/Akt signals were analyzed.The effect of AQP8 on the sensitivity of colorectal cancer cells to paclitaxel was also studied.Methods:Colorectal cancer cell lines SW480,HT-29,COLO 205,LoVo HCT116 and normal colorectal epithelial cells were purchased from Guangzhou Biotechnology Co.,Ltd.Grouping:SW480 and HT-29 cells were divided into control group,no-load group,AQP8 group,AQP8 + PI3K group,AQP8 + Akt group,shCon group,shPCDH7 group,AQP8 + PCDH7 group,paclitaxel group and AQP8 + paclitaxel group.The intervention dose of paclitaxel was 0.04 mol/L.CCK8 assay was used to detect the proliferation of breast cancer cells.Transwell assay was used to detect cell nvasiveness.Scratch test was used to detect the migration ability of cells.Apoptosis was detected by flow cytometry.Cell colony formation assay was used to detect the ability of cell colony formation.Western blot was used to detect the relative expression levels of PCDH7,PI3K and Akt protein after cell intervention in corresponding groups.Relative expression levels of AQP8,E-cadherin and N-cadherin were detected by RT-PCR.Result:1.The relative expression level of AQP8 in five colorectal cancer cells was significantly lower than that in human normal colorectal epithelial cells?P<0.05?.There was no significant difference in the relative expression level of miR-497 in five colorectal cancer cells?P>0.05?.2.In rectal cancer cells SW48 0 and HT-29,the relative expression level of AQP8 gene in AQP8 group was significantly higher than that in control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in the relative expression level of AQP8 gene between control group and no-load group?P>0.05?;the cell proliferation rate in AQP8 group was significantly lower than that in control group?P<0.05?and no-load group?P<0.05?,control group and no-load group?P<0.05?.There was no significant difference in reproductive multiple?P>0.05?;the number of perforating cells in AQP8 group was significantly lower than that in control group?P<0.05?and no-load group?P<0.05?,but there was no significant difference in the number of perforating cells between control group and no-load group?P>0.05?.The cell healing rate of AQP8 group was significantly lower than that of control group?P<0.05?and no-load group?P<0.05?.There was no significant difference between control group and no-load group?P>0.05?.The apoptotic rate of AQP8 group was significantly higher than that of control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in apoptotic rate between control group and no-load group?P>0.05?.The number of colony cells in AQP8 group was significantly lower than that in control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in the number of colony cells between control group and no-load group?P>0.05?.3.In two types of colorectal cancer cells?SW480 and HT-29 cells?,the relative expression levels of PI3K and Akt proteins in no-load group and AQP8 group were significantly different.The expression levels of PI3K and Akt protein in AQP8 group AQP8 cells were significantly lower than those without AQP8 group and AQP8+PI3K group?P<0.05?and AQP8+Akt group?P<0.05?s AQP8+PI3K group AQP8+PI3K group and AQP8+PI3K group AQP8+AQP8+AQP8+AQP8+Akt group had no significant difference(P>0.05?P>P>0.P>0.05?;the number of AQP8 group penepeneAQP8 cells was significantly lower than AQP8+PIPIPIPI3K8+PIPI3K8+PI3K8+PI3K8(P 8+PI3AQP8 + There was no significant difference in the number of perforating cells between Akt group and AQP8+ Akt group?P>0.05?;the healing rate of AQP8 group was significantly lower than that of control group?P<0.05?and no-load group?P<0.05?;the healing rate of AQP8 + PI3K group?P<0.05?and AQP8 + Akt group?P<0.05?was significantly higher than that of AQP8 + PI3K group and AQP8 + Akt group?P<0.05?.There was no significant difference in the number of colony cells?P>0.05?.4.In rectal cancer cells SW48 0 and HT-29,the relative expression level of PCDH7 protein in no-load group was significantly higher than that in AQP8 group?P<0.05?;the number of penetrating cells and wound healing rate in shPCDH7 group were significantly lower than those in control group and shCon group,while there was no significant difference in the number of penetrating cells and wound healing rate between control group and shCon group;the number of penetrating cells and wound healing rate in AQP8 + PCDH7 group were significantly higher than that in AQP group.The 8 group.5.The relative expression level of E-cadherin in AQP8 cells was significantly higher than that in control group,no-load group and AQP8+PCDH7 group.The relative expression level of N-cadherin in AQP8 cells was significantly lower than that in control group,no-load group and AQP8+PCDH7 group.6.In rectal cancer cells SW48 0 and HT-29,the number of colony cells in the control group was significantly higher than that in the paclitaxel group?P<0.05?and AQP8 + paclitaxel group?P<0.05?,and that in the paclitaxel group was significantly higher than that in the AQP8 + paclitaxel group?P<0.05?;the number of transmembrane cells in the control group was significantly higher than that in the paclitaxel group?P<0.05?and the AQP8 + paclitaxel group?P<0.05?,and that in the paclitaxel group was significantly higher than that in the AQP8 + paclitaxel group(P<0 The wound healing rate in control group was significantly higher than that in paclitaxel group?P<0.05?and AQP8 + paclitaxel group?P<0.05?,while that in paclitaxel group was significantly higher than that in AQP8 + paclitaxel group?P<0.05?.CONCLUSION:Overexpression of AQP8 can inhibit the proliferation,invasion and migration of colorectal cancer cells and promote cell apoptosis.It can also inhibit the expression of PI3K,Akt and PCDH7 in colorectal cancer cells.Overexpression of PI3K,Akt and PCDH7 can reverse the effect of overexpression of AQP8 on the biological function of colorectal cancer cells.Finally,overexpression of AQP8 increases the sensitivity of colorectal cancer cells to taxol.Part ?:In vitro study of the role of AQP8 in colorectal cancerAIM:To further study the effect of AQP8 on survival rate,tumor size and metastasis of transplanted colorectal cancer cells in nude mice.Methods:Sixty 4-week-old Balb/c male nude mice were divided into six groups,10 in each group,namely SW480-control group,SW480-no-load group,SW480-AQP8 group,HT29-control group,HT29-no-load group and HT29-AQP8 group.Subcutaneous tumorigenesis model:All kinds of cells were inoculated subcutaneously on the back of nude mice in each group at a dose of 0.2 ml per mouse.Hematogenous metastasis model:All kinds of colorectal cancer cell lines were digested routinely,and cell suspension concentration was adjusted to 5 X106/ml.0.1 ml cell suspension was injected into nude mice through tail vein.HE staining was used to make pathological sections of metastases.TUNEL was used to detect the apoptosis of cancer cells.Western blot was used to detect the expression of AQP8,PCDH7 and Akt in tissues.Result:The results of subcutaneous tumorigenesis model:1.In nude mice with transplanted colorectal cancer cells SW48 0 and HT-29,the relative expression level of AQP8 protein in AQP8 group was significantly higher than that in control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in the relative expression level of AQP8 protein between control group and no-load group?P>0.05?.2.In nude mice transplanted with colorectal cancer cell SW480,2 mice died in the control group,3 in the no-load group and 1 in the AQP8 group.In nude mice transplanted with colorectal cancer cell HT-29,the mortality rate was 25%in control group and no-load group,and no mice died in AQP8 group.There was no significant difference in mortality between the two groups?P>0.05?.3.In SW480 nude mice,the tumor volume?cm3?of control group,no-load group and AQP8 group were 3.57?+0.36?,3.84?+0.22?and 1.85?+0.17?,respectively.In HT-29 transplanted nude mice,the tumor volume?cm3?of control group,no-load group and AQP8 group were 3.00?+0.44?,3.72?+0.46?and 1.23?+0.19?,respectively.The tumour volume of AQP8 group was significantly smaller than that of control group?P<0.05?and no-load group?P<0.05?.There was no significant difference between control group and no-load group?P>0.05?.4.In the nude mice with SW480 transplanted tumors,the apoptotic rates?%?in the control group,the no-load group and the AQP8 group were 4.67?+2.75?,6.87?+6.33?and 45.01?+21.69?,respectively.In HT-29 transplanted nude mice,the apoptotic rates of tumor tissues in control group,no-load group and AQP8 group were 6.65?+3.23?,5.75?+3.39?and 44.67?+12.80?,respectively.The apoptotic rate of tumor tissue in AQP8 groupwas significantly higher than that in control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in apoptotic rate between control group and no-load group?P>0.05?.5.The expression levels of PCDH7 and Akt in tumors of nude mice transplanted with two types of colorectal cancer cells?SW48 0 and HT-29 cells?were significantly lower in AQP8 group than in control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in the expression levels of PCDH7 and Akt between control group and no-load group?P>0.05?.Hematogenous metastasis model:1 In nude mice with hematogenous metastasis of colorectal cancer cell SW480,the mortality of control group was 37.5%,that of no-load group was 62.5%,and that of AQP8 group was 12.5%.In nude mice with hematogenous metastasis of colorectal cancer cell HT-29,the mortality rate of control group was 25.0%,that of no-load group was 50%,and that of AQP8 group was not.The mortality rate of each group was significantly different?P<0.05?.2.In nude mice with hematogenous metastasis of colorectal cancer cell SW480,the mean deviations of metastases in control group,no-load group and AQP8 group were 6.63₁.85,7.63₃.11 and 1.63₁.30,respectively.In nude mice with hematogenous metastasis of colorectal cancer cell HT-29,the mean deviations of total metastases in control group,no-load group and AQP8 group were 7.38,3.02,8.75,2.25 and 1.88,1.36,respectively.The total number of metastases in AQP8 group was significantly less than that in control group?P<0.05?and no-load group?P<0.05?.There was no significant difference in the total number of metastases between control group and no-load group?P>0.05?.CONCLUSION:AQP8 can inhibit the growth of nude mice tumor tissue,promote cell apoptosis,reduce the mortality of nude mice and inhibit the metastasis of tumors.