The Role Of PPAPDC1A In Proliferation,Invasion And Metastasis Of Colorectal Cancer Cells

BackgroundColorectal cancer is the third most common malignant tumor in the world.Invasion and metastasis are the main causes of death.In recent years,with the continuous development of scientific research,early screening,early diagnosis and improved treatment,tumor mortality has been reduced,but the number of patients is still increasing.Phosphatidic acid phosphatase type 2 domain containing 1A(PPAPDC1A),is a new member of the phosphatidic acid phosphatase(PAP)family,also known as phosphophosphatase,can catalyze the dephosphorylation of multiple phosphates,which may interact with cell signaling is closely related.The study found that PPAPDC1 A is closely related to tumor development in lung cancer,breast cancer,cervical cancer,and liver cancer,but it is unclear whether PPAPDC1 A is related to the occurrence and development of colorectal cancer.ObjectivesColorectal cancer cell lines SW480,SW620,RKO,HCT116,DLD1 and LOVO were used as research objects to analyze the expression of PPAPDC1 A in different colorectal cancer cell lines.The effect of PPAPDC1 A on the proliferation,invasion and metastasis of six colorectal cancer cell lines was studied in vitro and in vivo.Methods1.To detect the expression of PPAPDC1 A in colorectal cancer paraffin samples by immunohistochemistry(IHC),and to analyze the relationship between PPAPDC1 A expression and clinicopathological parameters such as tumor differentiation,staging,metastasis and clinical prognosis.2.The expression of PPAPDC1 A m RNA and protein in colorectal cancer cells was detected by real-time quantitative PCR(q RT-PCR)and Western blot.3.To establish a stable overexpression and interference of colorectal cancer cell lines with PPAPDC1 A,and to detect the expression of PPAPDC1 A by q RT-PCR and Western blot.4.The effect of PPAPDC1 A on the proliferation of colorectal cancer cell lines was detected by CCK-8,plate clone formation and subcutaneous tumor formation in nude mice.5.The effects of PPAPDC1 A on the invasion,migration and metastasis of colorectal cancer cells were detected by transwell,scratch healing assay and nude mouse model of lung metastasis.Results1.The expression of PPAPDC1 A in colorectal cancer tissues: IHC results showed that the positive expression of PPAPDC1 A was mainly located in the cell membrane and part of the cytoplasm,showing brownish red or brownish yellow.In 73.3%(44/60)cases of colorectal cancer,the expression of PPAPDC1 A protein in tumor tissue was higher than that of matched normal colorectal tissue.The relationship between the expression level of PPAPDC1 A and clinicopathological parameters: The results showed that the expression level of PPAPDC1 A was not significantly different among different age groups,genders,tumor size,metastasis and non-metastatic colorectal cancer(P>0.05).The expression of PPAPDC1 A in colorectal cancers with different degrees of differentiation was statistically significant(P<0.05)and there was a negative correlation between them.2.The expression of PPAPDC1 A in colorectal cancer cell lines: The results of Western blot and q RT-PCR showed that PPAPDC1 A was differentially expressed in 6kinds of colorectal cancer cells(P<0.05).We selected relatively high expression of LOVO and SW620 cells and relatively low expression of RKO and SW480 cells forfollow-up experiments.3.Construction of lentiviral vector over-expressing and interfering with PPAPDC1 A and establishment of stably expressing cell line: Over-expression and interference lentiviral vector was constructed by molecular cloning technology and sequenced.The results showed that the inserted sequence was correct,no mutation and deletion or frame shift.Successful transfection of PPAPDC1A-overexpressing cell lines SW480-PPAPDC1 A,SW480-Vector;RKO-PPAPDC1 A,RKO-Vector;and PPAPDC1 A stable interference cell lines SW620-sh PPAPDC1 A,SW620-NC;LOVO-sh PPAPDC1 A,LOVO-NC were successfully performed using gene transfection technology.Successfully constructed cell lines were verified by q RT-PCR and Western blot.4.The effect of PPAPDC1 A over-expression and interference on the proliferation of colorectal cancer cells: The results of CCK-8 showed that the growth of PPAPDC1 A stable over-expression cell line was significantly faster than that of the control group(P<0.05);PPAPDC1A interfered cell lines grew significantly slower than the control group(P<0.05).The results of plate clone formation showed that compared with the control group,the number of clones over-expressed by PPAPDC1 A over-expression cells was significantly increased,the clonal area was significantly increased,the number of clones formed by PPAPDC1 A interference cell lines was significantly less than that of the control group,the clonal area was significantly reduced Small(P<0.05).The results of subcutaneous tumor formation in nude mice showed that the tumor growth rate was higher in PPAPDC1 A over-expression group than in control group(P<0.05),and the tumor growth rate was slower and the tumor volume was smaller in PPAPDC1 A interference group(P<0.05).The positive rate of Ki-67(75%)in PPAPDC1 A over-expression group was significantly higher than that in control group(35%)(P<0.05).The positive rate of Ki-67 in PPAPDC1 A interference group was significantly lower than that in control group(76%)(P<0.05).5.Effects of PPAPDC1 A over-expression and interference on invasion,migrationand metastasis of colorectal cancer cells: The results of transwell cell invasion assay showed that the number of cells stably crossing the basement membrane of PPAPDC1 A cells was significantly higher than that of the control group(P<0.05).However,the number of cells with PPAPDC1 A interfered by cell membrane was significantly less than that of the control group(P<0.05).Scratch test results showed that compared with the control group PPAPDC1 A stable over-expression of cell migration rate was significantly faster;and PPAPDC1 A interference cell line migration was significantly slower than the control group.The results of lung metastasis model in nude mice showed that the number of lung metastasis nude mice and the number of lung tumors in PPAPDC1 A stable over-expression cells were significantly larger than those in control group(P<0.05),while those in PPAPDC1 A interference cells the number of lung metastasis nude mice and the number of lung tumors was significantly less than the control group(P<0.05).Conclusions1.The expression of PPAPDC1 A in colorectal cancer tissues was significantly up-regulated.The expression of PPAPDC1 A was negatively correlated with the tumor differentiation.2.Over-expression of PPAPDC1 A promoted the proliferation,invasion,migration and metastasis of colorectal cancer cells,while PPAPDC1 A inhibited tumor cell proliferation,invasion,migration and metastasis after being interfered.