| Background and PurposeThe incidence rate and mortality rate of colon cancer are the top five in China and in the whole world.Colon cancer mainly occurs in people over 50 years-old,but in recent years,the incidence of colon cancer has been on the rise in people under 45.In recent years,with the increase of screening for the elderly,the progress of colonoscopy technology and surgical technology,the gradual popularization of MDT and the availability of a variety of targeted drugs,the prognosis of colon cancer patients has improved,but there is still a lack of biomarkers with good sensitivity and specificity.We should continue to explore the molecular mechanism in the occurrence,progress and metastasis of colon cancer,in order to find more sensitive markers for early diagnosis,prognosis and more effective therapeutic targets.In recent years,the G protein signal regulation(RGS)protein family has gradually become a hot topic in cancer research.At present,the mechanism of RGS16 in the occurrence and development of breast cancer,glioma and lung cancer has been reported,.However,the role of RGS 16 in colon cancer is rarely reported,and its molecular mechanism has not been reported yet.Methods1.Based on bioinformatics methods,the colon cancer related gene RGS 16 was screened from TCGA database,the expression level of RGS 16 was analyzed,and the correlation with different pathological types,disease stages and different degrees of tumor metastasis of colon cancer was analyzed,and the prognosis was analyzed.2.Paired cancer and adjacent tissues of 63 patients undergoing colon cancer resection in Shandong provincial hospital were included.The expression of RGS 16 was verified by immunohistochemical staining.The expression levels of RGS 16 in colon cancer and paired normal intestinal mucosa were detected by real-time quantitative PCR and Western blot.3.To study the effect of RGS16 on the biological function of colon cancer cells in vitro:the effects of RGS16 knockdown and overexpression on the proliferation of colon cancer cells were analyzed by cell proliferation test(MTT),cell clone formation test and edu test;The effects of RGS16 knockdown and overexpression on colon cancer cell cycle were analyzed by cell cycle detection.The effects of RGS 16 knockdown and overexpression on colon cancer cell apoptosis were analyzed by apoptosis experiment(annexin V-FITC/PI staining).The expression levels of apoptosis related proteins PARP,Bax and Bcl-2 in colon cancer cells after knockdown and overexpression of RGS 16 were detected by Western blot;The effects of RGS 16 knockdown and overexpression on the migration ability of colon cancer cells were analyzed by Transwell migration test and scratch test,and the expression levels of migration related proteins E-cadherin,N-cadherin and vimentin in colon cancer cells after knockdown and overexpression of RGS 16 were detected by Western blot.4.In vivo experiments were conducted to verify the effect of RGS 16 expression on colon cancer proliferation and apoptosis:the expression of RGS 16 in colon cancer cell line HT-29 was stably knocked down by lentivirus sh-rgs16 transfection,the proliferation model of subcutaneous tumor transplantation in nude mice was constructed,the volume and weight of subcutaneous tumor in knockdown group and control group were measured,and the growth curve was drawn,The apoptosis rate was determined by immunohistochemical staining of Ki67 and tunnel experiment.5.Second generation sequencing technology and bioinformatics methods were used to speculate the downstream signal pathway that can be regulated by RGS 16,and QRT PCR and Western blot were used to detect the expression level of related proteins in the target pathway in RGS 16 knockdown and overexpression cell lines,and then confirmed by rescue experiment,Whether up regulating the key protein VEGFR2 of the target signal pathway can reverse the changes of biological function of colon cancer cells caused by RGS 16 knockdown,including CCK-8 experiment,clone formation experiment,Transwell Migration Experiment and edu experiment.The above conclusions were further verified by immunohistochemical staining of the expression level of key proteins of the target pathway in the subcutaneous tumor tissue sections of the two groups of nude mice.Results1.Expression and clinical significance of RGS16 in colon cancer tissue by Shengxin analysis and clinical specimen study:(1)The results of TCGA analysis showed that the expression level of RGS16 was significantly up-regulated in colon cancer,and the expression level increased with the increase of disease stage and tumor metastasis.(2)Real time quantitative PCR and Western blot showed that the expression level of RGS16 in colon cancer tissues was significantly higher than that in adjacent normal tissues(P<0.05).(3)Immunohistochemical staining showed that the expression level of RGS16 in colon cancer was significantly higher than that in adjacent tissues.Chisquare test showed that the expression level of RGS16 was significantly correlated with TNM stage and distant lymph node metastasis of colon cancer(P<0.05),but not with age,gender,distribution and pathological differentiation(P>0.05).2.In vitro experiment to verify the effect of RGS16 on the biological function of colon cancer cells:(1)Three colon cancer cell lines with high/low expression of RGS16 were constructed.The results of Western blot showed that the expression level of RGS16 protein in HT29 and SW480 cell lines decreased significantly(P<0.01),while the expression level of RGS16 protein in HCT116 cell line increased significantly(P<0.01).(2)The results of MTT test,plate clone formation test and edu test showed that the up-regulated expression of RGS16 could significantly promote the proliferation of colon cancer cells,while the down-regulated expression of RGS16 could inhibit the proliferation of colon cancer cells.The difference between the groups was statistically significant(P<0.05,P<0.01,P<0.001,P<0.0001).(3)The results of cell cycle detection showed that in HT29 and SW480 cell lines with down-regulated RGS16,the proportion of cells in G0/G1 phase increased,while the proportion of cells in S phase decreased significantly,the difference was statistically significant.In HCT116 cell lines with up-regulated RGS16 expression,the proportion of cells in G0/G1 phase and G2/M phase decreased significantly,while the proportion of cells in S phase increased significantly,the difference was statistically significant(p<0.05,p<0.01,p<0.001,p<0.0001)?(4)The results of apoptosis experiment showed that the apoptosis rate increased significantly in HT29 and SW480 colon cancer cell lines knocking down RGS16(P<0.05,P<0.01,P<0.001,P<0.0001);on the contrary,the apoptosis rate decreased significantly in HCT116 cell lines overexpressing RGS16(P<0.05)Western blot analysis of apoptosis related proteins showed that after RGS16 expression was down regulated,the expression levels of Bax and PARP increased significantly(P<0.001),while the expression level of Bcl-2 decreased significantly(P<0.001);after RGS 16 expression was up regulated,the expression levels of Bax and PARP decreased significantly(P<0.001),while the expression level of Bcl-2 increased significantly(P<0.001).(5)Transwell migration experiment showed that the migration ability of colon cancer cells decreased significantly after the down-regulation of RGS 16 expression(P<0.05,P<0.01,P<0.001),while the migration ability of cells increased significantly after the overexpression of RGS 16(P<0.05)The scratch test showed that the cell migration rate decreased significantly after knockdown of RGS 16(P<0.05,P<0.01,P<0.001),while the cell migration rate increased significantly after overexpression of RGS 16(P<0.05)Western blot detection of migration related proteins showed that after knockdown of RGS 16 in HT29 and SW480 cell lines,the expression level of E-cadherin protein increased significantly,while the expression level of N-cadherin and vimentin decreased,the difference was statistically significant(P<0.05,P<0.01)In HCT116 cell line overexpressing RGS16,the expression level of E-cadherin protein decreased significantly,while N-cadherin and vimentin increased,the difference was statistically significant(P<0.01),suggesting that RGS 16 can promote the down-regulation of the expression level of E-cadherin and up-regulation of the expression level of N-cadherin and vimentin,so as to promote the invasion and migration of colon cancer.3.In vivo animal experiments verify the effect of RGS 16 on the proliferation and apoptosis of colon cancer cells.The proliferation model experiment of subcutaneous tumor transplantation in nude mice shows that:(1)After RGS 16 knockdown,the growth of subcutaneous tumors in nude mice slowed down and the volume of tumors decreased significantly(P<0.05,P<0.01).(2)Ki-67 staining of subcutaneous tumor tissue sections of nude mice:the positive proportion of stained cells in RGS 16 knockdown group was significantly lower than that in the control group(P<0.05).(3)Tunnel experiment showed that the apoptosis rate of RGS16 knockdown group increased significantly(P<0.05).4.Study on the mechanism of RGS16 promoting colon cancer proliferation and migration by regulating VEGFR2-PLCG2-PIK3CA-SPHK1-MAP2K1-ERK1 signal pathway.(1)Through second-generation sequencing and bioinformatics analysis,it is speculated that RGS16 may regulate the occurrence and development of colon cancer through VEGFR2-PLCG2-PIK3CA-SPHK1-MAP2K1-ERK1/2 signal pathway.(2)After RGS16 was stably knocked down by HT29 and SW480 cell lines,the transcription and expression levels of six key factors in the signal pathway,VEGFR2,Plcg2,PIK3CA,SphKl,map2kl and ERK1,were detected by qRT-PCR and Western blot.The results showed that they were down regulated in varying degrees,and the difference was statistically significant(P<0.05,P<0.01).The expression levels of four key proteins VEGFR2,PLCG2,PIK3CA and SPHK1 of VEGFR2 related signal pathway after RGS16 inhibition were verified in subcutaneous tumor tissues of nude mice.The knockdown group decreased significantly.(3)Rescue experiment:the expression level of VEGFR2 was up-regulated in two knockdown cell lines,and CCK-8 experiment,clone formation experiment,Transwell Migration Experiment and edu experiment were carried out.The results showed that the cell proliferation and migration ability of si-rgs16 knockdown group was significantly lower than that of the control group.After up regulating VEGFR2,the key protein of the target signal pathway,It can effectively reverse the decline of proliferation and migration of colon cancer cells caused by knockdown of RGS16,and achieve the expected rescue effect.Conclusions1.RGS16 is highly expressed in colon cancer and is associated with clinicopathologic features and poor prognosis.2.In vitro experiments showed that the down-regulation of RGS16 expression could inhibit the proliferation and migration of colon cancer cells,promote apoptosis and induce cell cycle arrest.Up regulating the expression level of RGS16 can promote the proliferation and migration of colon cancer cells and inhibit apoptosis.3.Animal experiments verify that down regulating the expression level of RGS16 can inhibit the proliferation of colon cancer cells and induce apoptosis in vivo.4.RGS16 can promote the proliferation and migration of colon cancer through VEGFR2-PLCG2-PIK3CA-SPHK1-MAP2K1-ERK1 signal pathway.5.RGS16 may become a new diagnostic and prognostic marker and molecular targeted therapeutic target for colon cancer. |
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