| Gastrointestinal tumors refer to tumors that originate in the digestive tract,including esophageal cancer,gastric cancer,colorectal cancer and liver cancer.The occurrence of these tumors is often closely related to people’s daily life and eating habits.Gastrointestinal tumors often present an insidious onset,and the patient is often diagnosed in the middle and advanced stages,and the mortality rate is higher than that of other malignant tumors.According to the latest global cancer burden data released by the International Agency for Research on Cancer of the World Health Organization in 2020,there are 19.29 million new cancer cases worldwide in 2020,including 1.09 million new cases of gastric cancer in 2020,ranking fifth in cancer incidence and 1.93 million new cases of colorectal cancer,ranking third in cancer incidence in 2020.And there were 9.96 million cancer deaths,including 770,000 deaths from gastric cancer,ranking fourth in cancer mortality and 930,000 deaths from colorectal cancer,ranking second among cancer deaths.The UBQLN family is a family of protein transporters.The family contains five structurally similar members,namely UBQLN1,UBQLN2,UBQLN3,UBQLN4,and UBQLNL.Relying on their shared UBA-UBL domain,the family members can mediate protein degradation.Studies have found that the aberrant expression of UBQLN family’s members can cause disturbances in protein homeostasis,leading to a series of diseases including neurodegenerative diseases and tumors.The current research on members of this family mainly focuses on their role in the occurrence and development of Alzheimer’s disease.There is no research report on UBQLN 1 in gastric cancer and colorectal cancer.In this study,GEO database mining was used to find that UBQLN1’s expression level of gastric cancer patients’ tissue samples was significantly higher than that of normal control people.Next,we used the lentiviral packaging system to construct gastric cancer cell lines AGS-UBQLN1,HGC27-UBQLN1 and its control cell lines AGS-CTRL and HGC27-CTRL that stably overexpress UBQLN1;and the UBQLN1 knock down gastric cancer cell line HGC27-shUBQLN1.1、HGC27-shUBQLN1.3 and its control cell line HGC27-shCTRL.In vitro experiments showed that enhanced UBQLN 1 can dramatically facilitate the proliferation,colony formation and epithelial-mesenchymal transition of gastric cancer cells,and at the same time increase the percentage of gastric cancer cells in the G2/M phase and accelerate the cell cycle process;conversely,knockdown of UBQLN1 can not only inhibit the proliferation,colony formation and epithelial-mesenchymal transition of gastric cancer cells,but also significantly reduce the percentage of cells in the G2/M phase of gastric cancer cells and slow down the cell cycle process.The results of in vivo experiments showed that knock down of UBQLN1 can inhibit the tumorigenesis and metastasis ability of HGC27 cells in nude mice.Furthermore,Western blot was performed to find that after knock down of UBQLN1 in HGC27,the expression level of cyclin-dependent kinase CDK1 was decreased,suggesting that UBQLN1 may regulate cell cycle changes by regulating the expression of CDK1.We also found that after overexpression UBQLN1 in gastric cancer cells AGS and HGC27,the phosphorylation level of ERK1/2 was increased significantly,and after knock down of UBQLN1 in HGC27,the phosphorylation level of ERK1/2 was decreased significantly,suggesting that UBQLN1 can regulate the ERK-MAPK pathway.We used ERK1/2 phosphorylation inhibitor U0126 to treat HGC27 cells overexpressing UBQLN1 at an appropriate concentration and found that the enhanced epithelial-mesenchymal transition ability of HGC27-UBQLN1 cells was restored to a certain extent,indicating that gastric cancer cells’ epithelial-mesenchymal transition could be modulated through regulation of ERK-MAPK pathway by UBQLN1.We also found that enhanced UBQLN1 in HGC27 caused observably increase of the protein level of β-catenin,the very core effective molecule of the Wnt/β-catenin pathway.On the contrary,after knocking down UBQLN1 in HGC27,the protein level of β-catenin was significantly reduced,suggesting that UBQLN1 can modulate the Wnt/β-catenin pathway in gastric cancer cells.Moreover,we also found that the expression level of UBQLN1 in colorectal cancer patients’ tissue samples was significantly higher than that of healthy people through GEO database.Furthermore,lnCAR database was used in prognosis analysis between UBQLN1’s expression level and survival.And we found that the colorectal cancer patients with high-level UBQLN1 expression had markedly lower overall survival and progression-free survival than colorectal cancer patients with low UBQLN1 expression levels,indicating that the expression level of UBQLN1 is negatively correlated with prognosis of colorectal cancer patients.Next,we used the lentivirus packaging system to construct the colorectal cancer cell line DLD1-UBQLN1 that stably over-expresses UBQLN1 and its control cell line DLD1-CTRL,and the colorectal cancer cell lines LoVo-shUBQLN1.1,shUBQLN1.2 that stably knock down UBQLN1 and its control cell line LoVo-shCTRL.In vitro experiments have shown that over expression of UBQLN1 dramatically encouraged the proliferation,colony formation and epithelial-mesenchymal transition of colorectal cancer cells,and it can also expand the percentage of cells in the G2/M phase of colorectal cancer cells and accelerate the cell cycle process;Conversely,knock down of UBQLN1 can not only lead to suppression of the proliferation,colony formation and epithelial-mesenchymal transition of colorectal cancer cells,but also markedly reduction of the percentage of cells in the G2/M phase of colorectal cancer cells and slow down the cell cycle process.In addition,we also found that knock down of UBQLN1 can down-regulate the anti-apoptotic protein SURVIVIN,but does not affect the level of colorectal cancer cells’ apoptosis;knock down of UBQLN1 can down-regulate the expression of LC3-II,indicating that knock down of UBQLN1 can inhibit colorectal cancer cells’ autophagy.The results of in vivo experiments showed that knock down of UBQLN 1 can inhibit the tumorigenesis and metastasis ability of LoVo cells in nude mice.Furthermore,we found that after knock down of UBQLN 1 in LoVo,the expression levels of Cyclin A2 and its dependent kinase CDK1 were decreased,suggesting that UBQLN 1 may regulate cell cycle through the expression of Cyclin A2 and CDK1.We also found after knock down of UBQLN1 in LoVo,the phosphorylation level of mTOR was increased,suggesting that UBQLN 1 may regulate cell’s autophagy through the classic mTOR pathway.Moreover,we found that after knock down of UBQLN1 in LoVo,the phosphorylation level of ERK1/2,MEK1 and the protein expression level of c-MYC were decreased,suggesting that knock down of UBQLN 1 can down-regulate the expression of ERK pathway and c-MYC.After we overexpressed c-Myc in colorectal cancer cells that knocked down UBQLN1,the proliferation ability of colorectal cancer cells was restored to a certain extent,indicating that UBQLN1 can regulate the proliferation of colorectal cancer cells by regulating the expression of c-Myc.In summary,this study clarified the role of UBQLN 1 in gastric cancer cells and colorectal cancer cells.In vitro experiments showed that UBQLN1 can promote the proliferation and EMT of gastric cancer cells and colorectal cancer cells,and accelerate the cell cycle process.In vivo experiments showed that knock down of UBQLN 1 can inhibit the tumor formation and metastasis ability of gastric cancer and colorectal cancer cells in nude mice.Furthermore,in gastric cancer cells,it was found that UBQLN 1 may act via regulation of the ERK-MAPK pathway and Wnt/β-catenin pathway;in colorectal cancer cells,it was also found that UBQLN1 may regulate autophagy in colorectal cancer cells through the classic mTOR pathway and down-regulate ERK pathway and c-MYC,and regulate the proliferation of colorectal cancer cells by regulating the expression of c-Myc.Our research is expected to point out the direction for further in-depth study of the mechanism of UBQLN1 in gastric cancer and colorectal cancer cells.Gastric cancer is one of the most common malignant tumors worldwide.According to the latest global cancer burden data released by the International Agency for Research on Cancer of the World Health Organization in 2020,there are 19.29 million new cancer cases worldwide in 2020,of which 1.09 million new cases of gastric cancer,ranking fifth in cancer incidence;global cancer deaths in 2 02 0 There were 9.96 million cases,including 770,000 deaths from gastric cancer,ranking fourth in cancer mortality.A large number of studies have shown that long non-coding RNAs plays an important role in the occurrence and development of many tumors.Long intergenic non-coding RNA 673(LINC00673),is firstly identified by Erica et al.,who using a genome-wide association study on pancreatic cancer cases;and they found the common variation of LINC00673 could enhance the susceptibility of pancreatic cancer.It’s also found that LINC00673 plays an important role in other types of tumors,such as non-small cell lung cancer,pancreatic cancer and breast cancer.However,there are few reports on LINC00673 in chronic gastritis and inflammatory response of gastric cancer.In this study,we noted enhanced expression of LINC00673 in gastric cancer samples as well as in gastritis samples,compared with normal samples.Furthermore,we used the Kaplan-Meier plotter database to find that the overall survival and post progression survival of gastric cancer patients with high LINC00673 expression levels were significantly lower than those of gastric cancer patients with low LINC00673 expression levels,indicating that the expression level of LINC00673 and the prognosis of gastric cancer patients are negatively correlated.We next used the lentiviral packaging system to construct gastric cancer cell lines AGS-LINC00673,HGC27-LINC00673 that stably overexpressed LINC00673 and their control cell lines AGS-CTRL,HGC27-LINC00673.In vitro experiments showed that overexpression of LINC00673 can significantly promote the proliferation,colony formation and migration of gastric cancer cells;it can also increase the percentage of gastric cancer cells in the G2/M phase and accelerate the cell cycle process.In vivo experiments showed that overexpression of LINC00673 can promote the tumorigenesis ability of HGC27 cells in nude mice.Nextly,RNA sequencing was performed using HGC27-LINC00673 and HGC27-CTRL cells,and the results were analyzed by GSEA(Gene set enrichment analysis),suggesting that the expression level of LINC00673 may be related to gastric cancer cell’s inflammation.To further validate our speculation,quantitative RT-PCR was performed to determine NLRP3 and IL-8 mRNA expression.We found that mRNA level of NLRP3 and IL-8 was high-expressed in AGS-LINC00673 and HGC27-LINC00673 cells compared to control ones respectively,suggesting enhanced LINC00673 promotes gastric cancer cell’s inflammatory response.To specify the specific mechanism by which LINC00673 overexpression promotes gastric cancer cell’s inflammatory response,We performed GO enrichment analysis on the RNA sequencing results and found that the Wnt/β-catenin pathway may be a potential way for LINC00673 to regulate the inflammatory response of gastric cancer cells.qPCR was performed to detect the mRNA level of LINC00673 and β-catenin in the serum samples of 36 patients with gastric cancer,and correlation analysis based on qPCR results found that the two were significantly positively correlated(correlation coefficient r=0.6704);In HGC27-LINC00673 cells,Western Blot and qPCR were performed to detect the protein level of β-catenin and the mRNA level of the downstream target gene set of the Wnt/β-catenin pathway.And the results showed that the protein level of β-catenin and the mRNA level of the target genes were significantly increased,after overexpression of LINC00673 in gastric cancer cell HGC27.The above results all indicate that LINC00673 may play a role in gastric cancer cells by regulating the Wnt/β-catenin pathway.Furthermore,ICG-001,a specific inhibitor of the Wnt/[3-catenin pathway,was used to perform rescue assay and it can restore the increased expression of inflammatory factors in LINC00673-overexpression cells,indicating that LINC00673 may regulate the inflammatory response of gastric cancer cells through Wnt/β-catenin pathway.The above studies show that LINC00673 is closely related to the occurrence and development of gastric cancer cells,especially the inflammatory response.Combined with the high expression of LINC00673 in the serum samples of patients with gastric cancer and gastritis,we found that the deviant expression of LINC00673 may be one of the molecular basis for the development of gastritis to gastric cancer,providing a new idea for the early diagnosis of gastric cancer. |
PDF Full Text Request