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Objective:To construct a nomogram model for predicting the progression-free survival (PFS) of patients with positive Rictor protein tested in lesion tissues of D2 + radical gastric adenocarcinoma resection and to analyze its predictive value. Methods:The tissue samples of 1 366 gastric adenocarcinoma patients who underwent radical resection in Shanxi Province Cancer Hospital from May 2005 to December 2020 were retrospectively collected, and the Rictor protein expression was detected by using the immunohistochemical SP method. The 676 Rictor-positive cases were grouped in a 7∶3 ratio by simple randomization, including 496 cases in the training cohort and 180 cases in the validation cohort. The correlation of Rictor protein expression and other clinicopathological factors with PFS of Rictor-positive patients was analyzed by using a multifactorial Cox proportional risk model to determine the independent influencing factors of PFS. The nomogram for predicting the 3-year and 5-year PFS rates of patients with gastric adenocarcinoma was constructed based on the independent influencing factors of PFS. The constructed nomogram was bootstrapped with 1 000 resamplings for internal validation to test the accuracy of the prediction model. The internal and external predictive efficacy of the model was further assessed by calibration curves, area under the curve (AUC) of time-dependent receiver operating characteristic (ROC) and decision curve analysis (DCA). The nomogram model was applied to score the PFS of 496 cases in the training cohort, and the X-tile software was used to obtain the best cut-off value for the score. The overall cohort, training cohort and validation cohort cases were divided into low-risk group (≤ best cut-off value) and high-risk group (> best cut-off value) according to the best cut-off value, and the Kaplan-Meier method was used to analyze the difference in PFS between the low-risk group and high-risk group.Results:Multifactorial Cox regression analysis showed that gender, age, pT stage, number of positive lymph nodes, neural invasion, tumor longest diameter, omental invasion, Clavien-Dindo classification of postoperative complications, and CGA expression were independent influencing factors for PFS of the training cohort with Rictor-positive gastric adenocarcinoma. The nomogram for predicting the 3-year and 5-year PFS rates of patients with Rictor-positive gastric adenocarcinoma was constructed based on the above indicators. The calibration curve for internal validation and external validation showed good agreement between the prediction of nomogram and actual PFS. The time-ROC curve showed that the AUC of the internally validated and externally validated models for predicting the 3-year PFS rate was 0.834 (95% CI 0.746-0.823) and 0.799 (95% CI 0.699-0.868), and the AUC for predicting the 5-year PFS rate was 0.817 (95% CI 0.718-0.821) and 0.795 (95% CI 0.675-0.895). The C index of the model for overall prediction was 0.795 (95% CI 0.764-0.825), which was better than the 8th edition of American Joint Committee on Cancer (AJCC) TNM staging [0.693 (95% CI 0.662-0.723)]; the external validation DCA showed that the C index of the model for prediction was 0.769 (95% CI 0.718-0.821). The X-tile software analysis showed that the best cut-off value for the PFS score of the training cohort model was 265.08, with 457, 337 and 120 cases in the low-risk group and 219, 159 and 60 cases in the high-risk group for the overall cohort, training cohort and validation cohort, respectively. Kaplan-Meier survival analysis showed that the median PFS time was not reached in the low-risk group for the overall cohort, training cohort and validation cohort, and the median PFS time was 24, 24 and 28 months in the high-risk group, and there were statistical differences in PFS between the low-risk and high-risk groups for each cohort (all P < 0.001). Conclusions:For the first time, a nomogram model for PFS prediction in gastric adenocarcinoma patients with Rictor-positive expression is successfully constructed, which could better distinguish between patients with low-risk and high-risk of PFS. For high-risk patients with Rictor-positive gastric adenocarcinoma, in addition to controlling tumor metastasis and postoperative complications, attention should be paid to the targeted therapy for positive expression of Rictor.
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OBJECTIVE@#To screen the microRNAs (miRNAs) targeting Rictor and investigate their effects in regulating the biological behaviors of colorectal cancer (CRC).@*METHODS@#Human colorectal cancer cell line KM12SM was transfected with the miRNAs targeting Rictor identified by prediction software to test inhibitory effects of these miRNAs on Rictor expression using qRT-PCR and Western blotting. Dual luciferase reporter assay was used to further confirm the binding of these miRNAs to the 3'UTR of Rictor mRNA. Cell survival and colony formation assays were used to investigate the effects of these miRNAs on survival and colony formation in KM12SM cells.@*RESULTS@#miR-152 and miR-448 were identified as the Rictor-targeting miRNAs, which significantly inhibited the expression of Rictor in KM12SM cells ( < 0.05). The two miRNAs were confirmed to bind to the 3'UTR of Rictor mRNA and significantly inhibited luciferase activity in KM12SM cells ( < 0.01, < 0.05); they also showed activities of posttranscriptional modulation of Rictor. Overexpression of miR-152 and miR-448 both significantly inhibited the growth and colony formation of KM12SM cells.@*CONCLUSIONS@#miR-152 and miR-448 can down-regulate the protein expression of Rictor by targeting Rictor mRNA to negatively regulate the growth and colony formation of colorectal cancer cells.
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Humans , 3' Untranslated Regions , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms , Drug Therapy , Gene Expression Regulation, Neoplastic , MicroRNAs , Pharmacology , Rapamycin-Insensitive Companion of mTOR ProteinABSTRACT
Objective: To investigate the effect of silencing rapamycin insensitive companion of mammalian target of rapamycin (RICTOR) gene expression on the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and to explore its possible molecular mechanism. Methods: The expression level of RICTOR protein in esophageal squamous cell carcinoma TE1, ECa109, EC9706, KYSE450 and KYSE790 cells were detected by Western blotting. RICTOR-shRNA or the Control-shRNA was transfected into ECa109 cells by LipofectAMINE, and the ECa109 cells stably expressing RICTOR-shRNA or the Control-shRNA were screened and named as ECa 109-RICTOR-shRNA or ECa109-control-shRNA cells. The effect of everolimus on the proliferation of ECa109-RICTOR-shRNA cells was detected by CCK-8 assay. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt)/mammalian target of rapamycin (mTOR) signal pathwayrelated RICTOR, Akt, phospho-Akt (p-Akt) (Ser473), ribosome protein subunit 6 kinase of 70 kDa (p70S6K), phospho-p70S6K (p-p70S6K), proline-rich Akt substrate of 40 kDa (PRAS40) and phospho-PRAS40 (p-PRAS40) (Thr246) proteins in everolimus-treated ECa109-control-shRNA and ECa109-RICTOR-shRNA cells were detected by Western blotting. The nude mouse xenograft tumor models of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells were established and treated with everolimus, then the effect of everolimus on tumor growth in nude mice was evaluated. Results: RICTOR protein was expressed in five esophageal squamous cell carcinoma cell lines, especially in ECa1 09 cells. Compared with the Control-shRNA, RICTOR-shRNA inhibited the proliferation of ECa1 09 cells. Everolimus inhibited the proliferation of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells, especially to the former; the values of half maximal inhibitory concentration (IC50) were (17.68± 1.25) μmol/L and (36.84±1.57) μmol/L, respectively. The RICTOR-shRNA decreased the expression levels of p-Akt (Ser473) and p-PRAS40 (Thr246) (both P 0.05), which indicated that RICTOR-shRNA inhibited the phosphorylated activation of Akt and PRAS40 induced by everolimus. Both RICTOR-shRNA and everolimus inhibited the growth of ECa109 cell xenografts in nude mice (all P < 0.05), while the inhibitory effect was strongest in RICTOR-shRNA+everolimus group (P < 0.001). Conclusion: Silencing RICTOR gene can improve the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and the molecular mechanism may be associated with the down-regulation of RICTOR expression to inhibit the phosphorylated activation of Akt and PRAS40 induced by everolimus.
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Objective To investigate the effect of resveratrol on the proliferation, migration and angiogenic ability of HUVECs mediated by Rictor over-expression adenovirus.Methods The Rictor was obtained through PCR and cloned into GV314 plasmid to construct recombinant plasmid, then co-transfected 293T cells with helper plasmids to obtain Rictor overexpressing adenoviral particles(Ad-Rictor),the vector without target gene Ad-Null was set as the negative control group.Ad-Rictor and Ad-Null were infected HUVECs respectively,we also set up blank control group and resveratrol-intervention group(Ad-Rictor+Res). The expression of recombinant protein was detected by fluorescence microscopy and Western blot. CCK-8 assay,wound healing and matrigel assay were performed to as-sess the proliferation,migration and tube formation of HUVECs. Results We constructed Ad-Rictor and Ad-Null which may infect HUVECs and express Rictor protein efficiently. Ad-Rictor could significantly improve the prolifer-ation,migration and lumen formation (P<0.05), resveratrol intervention may significantly inhibit these functions induced by Ad-Rictor (P<0.05). Conclusions Resveratrol inhibits the proliferation, migration and angiopoietic ability of HUVECs through targeting mTORC2/Rictor.
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Purpose To detect the expression of Raptor,Rictor,angiogenesis-related factors HIF-lα,HIF-2α and VEGF and to investigate their relationship and significance in eolorectal cancer (CRC).Methods Immunohistochemistry,Western blot and RT-PCR were employed to detect the expression of Raptor,Rictor,HIF-lα,HIF-2α and VEGF in 120 cases of CRC and 60 cases of normal colorectal mucosa.CD34 labeled microvascular density (MVD) was also observed.The correlations between Raptor,Rictor,HIF-1α,HIF-2c,VEGF expression and the patients' clinicopathological features were analyzed.Results The positive rates of Raptor,Rictor,HIF-1c,HIF-2α and VEGF in CRC were significantly higher than those in normal colorectal mucosa (P ＜ 0.05).Raptor and Rictor expression was correlated with the degree of tumor diffcrentiation and lymph node metastasis,respectively.The expression of HIF-1α,HIF2α and VEGF was higher in patients with lymph node metastasis than those in patients without lymph node metastasis (P ＜0.05).The MVD was higher in patients with Raptor or Rictor positive than that in patients with Raptor or Rictor negative (P ＜0.05).The expression of Raptor was positively correlated with HIF-1α and VEGF (P ＜ 0.01),the expression of Rictor was positively correlated with HIF-2o and VEGF (P ＜ 0.01),but the expression of Raptor was negatively correlated with Rictor (P＜0.01).Conclusion The expression of mTOR core molecules Raptor and Rictor is related to the initiation and development of colorectal cancer and angiogenesis,and they promote angiogenesis in colorectal cancer by different ways.
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Objective To construct recombinant lentivirus silence vector aiming at rictor gene in mTORC2 specific protein, and to investigate its regulation on mTORC2/SGK1 signal pathway and the effect on pulmonary alveolar epithelial sodium ion chan-nel,as well as the role in acute respiratory distress syndrome(ARDS)and acute lung injury.Methods The interfering vector plas-mid and empty vector plasmid of target gene rictor were constructed,which and the lentivirus packaging system were co-transfected to 293T cells.The viral supernatant was collected,centrifuged,concentrated and purified for obtaining recombinant lentivirus.The virus titer was detected and the virus was infected to A549 cells.Stable cell lines were screened.RT-PCR was used to confirm the silencing situation of target gene rictor.The expression situation of various signal indexes in this pathway was detected by PCR and Western blot.Results The recombinant lentivirus of silence gene rictor was successfully constructed and transfected to A549 cell for obtaining stable cell lines.Compared with blank and control groups,the mRNA levels of rictor,downstream SGK1 andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased (P <0.05 ).Meanwhile,the protein levels of rictor,downstream SGK1,P-SGK andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased compared with the other two groups(P<0.05).Conclusion Silence rictor gene has the obvious regulation effect on mTORC2/SGK1 signal pathway,meanwhile affects the expression of pulmonary alveolar epithelial cellular α-,β-and γ-ENaC at gene and protein level.It is speculated that mTORC2/SGK1 may be an important signal pathway for regulating the clearance capacity of pulmonary alveolar epithelial cells on pulmonary alveolar fluid and simultaneously affecting the pulmonary edema formation.
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Objective To study the role of RICTOR on rheumatoid arthritis-fibrobast-like synoviocytes (RA-FLS) activation.Methods FLS were isolated from the primary synovial tissues,which were obtained during joint replacement surgery or arthroscopy from three patients with RA.RA-FLS were stimulated with TNF-α at the dose of 10 ng/ml and 20 ng/ml for 48 h.The expression of RICTOR was detected by western blotting.Chemically synthesized RICTOR gene targeted for double-stranded siRNAs were transfected into RA-FLS by cationic liposome.After being transfected with RICTOR siRNA for 48 h,RA-FLS was treated with or without TNF-α for 48 h.The expression of RICTOR was evaluated by western blotting,and the cell viability was analyzed by methylthiazoltetrazolium (MTT) assay.The data were analyzed using analysis of variance (ANOVA) and LDL-t test.Results The expression of RICTOR protein was significantly higher in the TNF-α stimulated group (at the dose of 10 ng/ml and 20 ng/ml for 48 h) than that in the control group (bothP＜0.05),while the mean change of RICTOR/GAPDH ratios of band optical density x+s was 0.35±0.06 for the control group,0.60±0.09 for the TNF 10 ng/ml group and 1.10±0.12 for the TNF 20 ng/ml group.Moreover,the expression of RICTOR protein was obviously decreased in RICTOR siRNA transfection groupthan that in control after being trans-fected for 96 h (both P＜0.05),and ratios of control group,RICTOR (-)/TNF-α(-) group and RICTOR(-)/TNF-α(+) group was 0.498 4±0.140 1,0.012 8±0.002 0,0.042 5±0.027 3respectively.After the silence of RICTOR,the cell viability decreased in RA-FLS,no matter with or without TNF-α for 48 h later (both P＜0.05).Conclusion These results indicat that RICTOR might play an important role in the TNF-α associated activation of RA-FLS.
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Objective To investigate the different distribution and expression of mammalian target of rapamycin complex (mTORC) in the kidney of diabetic nephropathy (DN) mice.Methods Fourteen eight-week-old male C57BL/6 mice were assigned to 2 groups: the control group ( n=7 ) and the streptozotocin ( STZ )-induced DN group ( n=7 ) . Blood and urinary variables including glucose , albumin, creatinine and albumin/creatinine ratio were assessed 2 weeks after STZ injection.Hematoxylin-eosin staining was performed for renal pathological analyses .The distributions of mTOR , phosph-ser2448-mTOR(p-mTOR), mTORC1(Raptor), mTORC2(Rictor) and phosph-ser240/244-S6K1 (p-S6K1) were determined by immunofluorescence.The expression levels of mTOR, p-mTOR, mTORC1(Raptor), mTORC2(Rictor), S6K1 and p-S6K1 were detected by Western blotting .Results Two weeks after STZ injection , the diabetic mice developed albuminuria (P<0.01) and renal hypertrophy (P<0.05).The immunofluorescence positive staining for mTOR , Raptor, and Rictor was distributed in the epithelial cells of proximal tubules , glomerular mesangium and capillary loops as well as the medullary collecting ducts of the control mouse kidney .These positive signals increased in the DN mouse kidney ( P<0.05).However, pS6K1 was not detected in the inner medulla of control mouse and p-mTOR was not found in the glomeruli of both control and DN mice .Conclusion mTORC is widely expessed in the mouse kidney and participates in the development of DN , whereas the 2448 serine phosphorylation of mTOR may be not implicated in the hyperglycemia mediated glomerular injury .