CLDN6 Regulates P53 Through PTEN/Akt Pathway To Inhibit The Proliferation Of Colorectal Cancer Cells

The expression of tight junctions?TJs?protein CLDNs is abnormal in colorectal cancer and other epithelial cancer,which affects the proliferation of tumor through different molecular mechanisms.Claudin-6?CLDN6?is one of the members of CLDNs family.CLDN6 has an N-terminal,a C-terminal and two extracellular loop structures.Its C-terminal has a PDZ binding motif which binds to a PDZ domain-containing signaling protein?such as ZO-1,etc.?,and which is involved in the signal transmission process in cells.As a result,CLDN6 plays an important role in the regulation of tumor cell proliferation.Our previous work found that CLDN6 was underexpressed in colorectal cancer cells and overexpression of CLDN6 inhibited the proliferation of colorectal cancer cells.In addition,CLDN6 upregulated the expression of p53 to mediate chemotherapy resistance in MCF-7.At the same time,the pre-experimental results showed that CLDN6 up-regulated the expression of p53 in colorectal cancer cells.Therefore,we speculated that the inhibition of CLDN6 on the proliferation of colorectal cancer cells was related to the upregulation of p53 expression.p53 is a tumor suppressor gene.When cells are stimulated by ionizing radiation,carcinogens and mutagens,p53 protein binds to the corresponding binding sites of DNA and plays the role of transcription factors,which activates p21 gene transcription to block cell cycle and repair DNA.p53 induces apoptosis if the repair fails.However,mutation or loss of p53 cannot repair damaged DNA,causing abnormal proliferation of cells and development of malignant tumors.p53 is a short-lived protein and there are many factors regulating the stability of p53 protein in the body,among which MDM2 is the main negative regulator of p53 gene.MDM2can regulate a variety of functions of p53,including directly blocking the N-terminal transcription activation domain of p53,promoting the transformation of p53 from the nucleus to the cytoplasm,and mediating the ubiquitination and degradation of p53.MDM2 is a downstream target gene of Akt signaling pathway.Akt is a serine/threonine kinase that can specifically bind to serine or threonine residues of substrate proteins.The PH domain of Akt can combine with phosphatidylinositol-3-phosphate?PIP3?,which makes Akt transfer from the cytoplasm to the membrane and phosphorylated,thus activating the Akt signaling pathway.PTEN is a tumor suppressor gene with phosphatase activity and PIP3 is its important substrate.PTEN dephosphorylates PIP3 to generate PIP2 at the D3 position,thereby inhibiting the activation of the Akt signaling pathway.The C-terminus of PTEN has a PDZ binding motif.Therefore,we speculated that CLDN6 can bind to PTEN through the PDZ domain.Therefore,we speculate that CLDN6 regulates p53 through the PTEN/Akt signaling pathway to inhibit colorectal cancer cell proliferation.ObjectiveTo investigate the mechanism of CLDN6 regulating p53 through the PTEN/Akt signaling pathway to inhibit the proliferation of colorectal cancer cells,and to provide theoretical support and experimental evidence for the mechanism of CLDN6inhibiting the proliferation in colorectal cancer.Methods1.The expression of CLDN6 in colorectal cancerThe expression of CLDN6 in colorectal cancer patients was analyzed by GEO database.The GSE44076 data set contained 98 paired colorectal cancer and adjacent tissues,and the GDS4515 data set contained 34 colorectal cancer tissues and 15adjacent tissues;RT-PCR and Western-Blot were used to detect the expression of CLDN6 in colorectal cancer cell lines HCT116 p53^+/+and HCT116 p53^-/-.2.Effect of CLDN6 on the proliferation ability of colorectal cancer cells?1?Construction of stable transfected cell lines overexpressing CLDN6:CLDN6overexpressing plasmids and auxiliary plasmids were used in 293T cells to package lentiviruses overexpressing CLDN6,and lentiviruses were used to establish HCT116p53^+/+and HCT116 p53^-/-cell lines overexpressing CLDN6 by infecting cells;The transfected cells were identified by RT-PCR and Western-Blot,and we detected the location of CLDN6 by immunofluorescence.?2?The effect of CLDN6 on the proliferation of colorectal cancer cells:The effects of CLDN6 on the proliferation of colorectal cancer cells HCT116 p53^+/+and HCT116 p53^-/-were detected by CCK-8 and clone formation experiments.3.Mechanism of CLDN6 regulating p53 on colorectal cancer cell proliferationIn order to study the mechanism of the inhibition of cell proliferation by CLDN6,transfecting overexpressing CLDN6 and empty plasmids in HCT116 p53^+/+were used as research objects.?1?Regulation of CLDN6 on p53:RT-PCR and Western-Blot were used to detect the expression of p53.Cellular immunofluorescence and Western-Blot were used to detect the subcellular localization of p53.Actinomycin?CHX?was added during cell culture and Western-Blot was used to detect the half-life of p53.?2?CLDN6 regulates p53 expression through PTEN/Akt:Western-Blot was used to detect the expression of ZO-1 and PTEN;co-immunoprecipitation was used to detect the binding of ZO-1 and PTEN in the clone group;Western-Blot was used to detect the expression of PTEN,Akt and MDM2.PTEN inhibitor VO-Ohpic was added during cell culture.Western-Blot was used to detect the expression of PTEN,Akt and p53.Results1.Low expression of CLDN6 in colorectal cancerThe analysis of GEO database showed that CLDN6 was underexpressed in colorectal cancer tissues compared to adjacent tissues?P<0.01?;RT-PCR and Western-Blot results showed that the expression of CLDN6 in HCT116 p53^+/+and HCT116 p53^-/-cells was lower than that in positive control cells(HCT116p53^+/+P_mRNA<0.05,P_protein<0.01;HCT116 p53^-/-P_mRNA<0.05,P_protein<0.05).These results suggested that the expression of CLDN6 was low in colorectal cancer and CLDN6plays a tumor suppressive effect.2.CLDN6 depends on p53 to inhibit the proliferation of colorectal cancer cells?1?Identification of stable transgenic cell lines overexpressing CLDN6:By performing RT-PCR and Western-Blot,we found the expression of CLDN6 in clone group was higher than that in empty group(HCT116 p53^+/+P_mRNA<0.001,P_protein<0.05;HCT116 p53^-/-P_mRNA<0.01,P _protein<0.05);the results of immunofluorescence showed that CLDN6 was mainly distributed in cell membrane and cytoplasm.The results showed that we successfully constructed a cell line that overexpressed CLDN6.?2?The effect of CLDN6 on the proliferation of colorectal cancer cells:The results of CCK8 showed that the cell viability of HCT116 p53^+/+clone group was lower than that of the empty group?P<0.05?,and the number of clone formation of HCT116 p53^+/+clone group was lower than that of the empty group?P<0.05?.However,there was no significant change in cell proliferation and colony forming ability of HCT116 p53^-/-clone group.The results showed that CLDN6 inhibited colorectal cancer cell proliferation which was associated with p53.3.The mechanism of CLDN6 regulating p53 inhibiting cell proliferation?1?Regulation of CLDN6 on p53:By performing RT-PCR and Western-Blot,we found the expression of p53 in the clone group increased?P<0.05?,but there was no significant change in mRNA;the results of cell immunofluorescence and Western-Blot showed that the expression of p53 in the nucleus of the clone group increased compared with that of the empty group?P<0.05?;after treatment with CHX,the half-life of p53 in the clone group was prolonged?P<0.05?.?2?CLDN6 regulates p53 expression through PTEN/Akt:The results of co-immunoprecipitation showed that there was a combination of ZO-1 and PTEN protein in the clone group.By performing Western-Blot,we found the expression of PTEN in the clone group was higher?P<0.001?,and phosphorylated Akt and MDM2in the clone group were lower than in the empty group(P_Akt<0.01,P_MDM2<0.001);PTEN inhibitor reversed the regulation of p53 by CLDN6.Conclusion?1?CLDN6 is low-expressed in colorectal cancer?2?CLDN6 suppresses the proliferation of colorectal cancer through p53 in vitro?3?CLDN6 regulates the expression of p53 through the PTEN/Akt pathway...