The Function And Mechanism Of Piezo1 In The Progression Of Colon Cancer

According to the 2020 World Cancer Statistics report,colorectal cancer has become the third most common disease with 1931590 new cases and the incidence rate is 10.0%,ranking behind only breast and lung cancer,at the same time,it is also the second leading disease with the highest number of deaths.935173 patients have died,with a mortality rate of 9.4%,which is only lower than that of lung cancer.When most patients in our country go to the hospital for treatment because of their symptoms and signs,the State of cancer has already developed to the middle and late stage,and some even have already appeared extensive distant organ metastasis,this results in poor results of traditional surgical treatment and 5-year survival.With the deep research on the pathogenesis of Colon Cancer,the combination of signal transduction,cell receptor and other molecular targeted gene therapy and immunotherapy is based on the traditional drug therapy and surgery,radiotherapy and chemotherapy,it can significantly improve the quality of life and prolong the survival time of patients with colon cancer.Therefore,the search for more efficient and accurate diagnosis and treatment of colon cancer has become the focus of research at home and abroad.Piezo1 protein has been found to be highly expressed in many cancer tissues,such as Glioblastoma multiforme,bladder and prostate cancer,and is involved in the proliferation,invasion and metastasis of tumor cells.However,the role of Piezo1 in the pathogenesis of Colon cancer has not been reported.In order to investigate the effect of Piezo1 protein on the biological behavior of human colon cancer cells,human colon cancer tissues and human colon cancer cell lines HCT116 and SW480 were used as research objects,to explore the potential role of PIEZO1 and HIF-1 α/VEGF signal pathway in colon cancer cell metastasis,and to provide a new direction for early monitoring,screening,treatment and prognosis evaluation of colon cancer.This study is divided into three parts.PartⅠ Expression of piezo1 in colon cancer and its correlation with prognosisObjective: To observe the expression of Piezo1 in colon cancer and adjacent tissues at m RNA and protein levels,and to explore the relationship between Piezo1 expression and clinical characteristics,therapeutic effect and survival time of patients with colon cancer.Methods:1.The protein expression of Piezo1 and HIF-1α in well,poorly differentiated and adjacent tissues were observed by immunohistochemistry.2.The m RNA expression of Piezo1 and HIF-1α in poorly differentiated colon cancer and adjacent tissues were analyzed by q RT-PCR.3.The protein expression of Piezo1,HIF-1α and VEGF in poorly differentiated colon cancer and adjacent tissues were checked by Western-blot.4.Chi-square test was used to explore the correlation between Piezo1 expression and clinical characteristics of patients with colon cancer.5.Kaplan-Meier method was used to investigate the relationship between Piezo1 expression and therapeutic effect and survival time in patients with colon cancer.Results:1.The results of immunohistochemistry showed that,there were significant differences in the positive rates of Piezo1 and HIF-1α protein expression between adjacent tissues and colon cancer tissues with different degrees of differentiation(all P<0.05).The positive rates of Piezo1 and HIF-1α were all significantly higher than those in adjacent tissues in poorly differentiated colon cancer tissues.In well-differentiated colon cancer tissues,the positive rate of HIF-1α was significantly higher than that in adjacent tissues,while the change of Piezo1 was not significantly changed,indicating that the expression of Piezo1 was more specific in poorly differentiated colon cancer tissues.Low-differentiated colon cancer tissues were used in further studies.2.qRT-PCR data showed that the expressions of Piezo1 and HIF-1α at m RNA level in colon cancer tissue were all significantly higher than those in adjacent tissues(both P<0.05).3.Western-blot data showed that,expressions of Piezo1,HIF-1α and VEGF in colon cancer tissue were all significantly higher at the protein level than those in adjacent tissues(all P<0.05).4.Based on the median of Piezo1 protein level in colon cancer tissues,patients were divided into Piezo1 high expression group and Piezo1 low expression group.There were no significant differences in age,gender and TNM stage between Piezo1 high expression group and Piezo1 low expression group(all P>0.05).5.The survival rate of the high Piezo1 group was 21.2 %,and that of the low Piezo1 group was 25.4 %.The survival rate in Piezo1 high expression group was 4.2 % lower than that in Piezo1 low expression group(P<0.05).Conclusions:1.Expressions of Piezo1,HIF-1α and VEGF at protein level were all significantly increased in poorly differentiated colon cancer tissues.2.The expression level of Piezo1 may be related to the pathological type of colon cancer,and is rarely affected by the course of disease,gender and other factors.3.In patients with high expression of Piezo1 protein in poorly differentiated colon cancer tissues,the survival rates are significantly reduced and the prognoses are poor.Part Ⅱ Effects of Piezo1 on proliferating,invasion and migration of colon cancer cellsObjective: to investigate the role of Piezo1 on proliferation,invasion and migration in HCT116 and SW480 cellsMethods:1.Three specific Piezo si RNA were designed and the efficiency of si RNA knockdown of Piezo1 was detected by q RT-PCR and Western blot.2.CCK-8 was used to analyze the effects of Yoda1 on the on the proliferation of HCT116 and SW480 cells.3.The effect of Yoda1 on Piezo1 levels in HCT116 and SW480 cells was analyzed at the protein level by immunocytochemistry.4.Cell scratch and transwell experiment were used to check the effect of Piezo1 on migration and invasion of HCT116 and SW480 cellsResults:1.q RT-PCR and Western-blot data showed that,the expression of Piezo1 was significantly decreased in HCT116 and SW480 cells at m RNA and protein levels with si RNAs transfection(all P<0.05),among which,si RNA3 exhibited the best efficiency of Piezo1 knockdown and was used for following studies.2.Data from CCK-8 showed that,the increase of Yoda1 concentration inhibited the proliferation of HCT116 and SW480 cells(all P < 0.05).Yoda1 with small effect on proliferation and apoptosis of HCT116 and SW480 cells was selected for subsequent experimental study.3.Immunocytochemical data showed that,Piezo1 knockdown could significantly reduce the expression of Piezo1 protein in HCT116 and SW480 cells.After Yoda1 activation and opening Piezo1 ion channel,the Piezo1 levels in these two cells were significantly increased,and the differences were statistically significant(both P<0.05).4.Cell scratch and Transwell experiment indicated that,pizeo1 knockdown could inhibit cell migration and invasion,while Yoda1 treatment enhanced cell migration and invasion(all P<0.05).Conclusions:1.Piezo1 si RNA transfection could inhibit the expression of Piezo1 in HCT116 and SW480 cells,among which si RNA3 showed the best efficacy.and was selected for subsequent studies.2.Yoda1 decreased the proliferation of HCT116 and SW480 cells.The concentration of 50 μM Yoda1 was selected for subsequent experiments.3.Piezo1 knockdown can lead to significant decrease in cell migration and invasion ability,and activation of Piezo1 can increase the invasion and migration ability of HCT116 and SW480 cells.Part Ⅲ The mechanism underlying Piezo1 regulating metastasis of coloncancer cellsObjective: the investigated the effect of Piezo1 on HIF-1α and VEGF expression in HCT116 and SW480 cells,and to explore the important role of HIF-1α playing on Piezo1 effection.Methods:1.HIF-1α protein expression was detected by immunocytochemistry in HCT116 and SW480 cells with Piezo1 knockdown or Yoda1 treatment.2.the expressions of Piezo1,HIF-1α and VEGF were detected by Western-blot in HCT116 and SW480 cells with Piezo1 and HIF-1α silence.3.q RT-PCR was used to detect expressions of Piezo1,HIF-1α and VEGF were in HCT116 and SW480 cells with HIF-1α.silencing4.Transwell assay was used to determine the effect of Yoda1 on the invasion and migration of HCT116 and SW480 cells with HIF-1α silencing.Results:1.Immunocytochemical data showed that,compared with the control group,the expression of HIF-1α in HCT116 and SW480 cells was significantly decreased by Piezo1 knockdown while significantly increased by Yoda1 treatment(all P<0.05).2.Western blot results showed that,compared with the control group,Piezo1 knockdown could reduce the expressions of HIF-1α and VEGF significantly in HCT116 and SW480 cells(all P < 0.05).HIF-1α silencing could reduce the expression of VEGF significantly(P < 0.05).but caused no alteration in Piezo1 expression.3.q RT-PCR results showed that,compared with the control,Yoda1 treatment could not increase the expression of VEGF in HCT116 and SW480 cells with HIF-1α silencing(all P < 0.05).4.Transwell experiment data showed that,HIF-1α silencing could reduce the invasion ability of HCT116 and SW480 cells significantly(both P<0.05),and these could not be reversed by Yoda1 treatment(P > 0.05).Conclusions:1.Piezo1 activation can increase the expression of HIF-1α and VEGF.HIF-1α participates in the effects of Piezo1 on tumor cells and is a key factor of Piezo1 function.2.The activation of Piezo1 may promote the metastasis of colon cells through activation of HIF-1α / VEGF pathway.