| BackgroundGastric cancer(GC)is one of the most common types of malignant tumors,especially digestive system tumors.According to the latest national cancer statistics released by the National Cancer Center,the incidence rate and mortality of GC are among the top three malignant tumors in China.Gastric cancer is a disease with high molecular and phenotypic heterogeneity,strong invasion and metastasis,and short survival time.Surgery is the main means to treat gastric cancer,and abdominal metastasis is the main factor affecting the prognosis of gastric cancer patients.Because the onset of gastric cancer is relatively hidden,and there is still a lack of early specific diagnostic markers,most patients with gastric cancer in China are already in the stage of gastric cancer progression when they are first diagnosed,and many of them cannot undergo radical resection because of tumor metastasis.Even after radical resection of gastric tumor,a considerable proportion of patients were diagnosed with tumor recurrence or metastasis during postoperative treatment and follow-up,resulting in a low five-year survival rate and poor prognosis.Therefore,it is of great significance to explore the possible process and corresponding mechanism of gastric cancer metastasis to find new targets for precise treatment of gastric cancer patients and actively improve the prognosis.Calreticulin(CALR),located in the p13.2-p13.3 region of human chromosome 19,is a highly conserved endoplasmic reticulum calcium binding protein with a size of 46 KD.It is mainly distributed in the nuclear envelope and endoplasmic reticulum cavity of cells.For human beings,its existence is very common.Due to its special structure,recent studies have shown that CALR has many biological functions,such as regulating calcium balance,participating in antigen presentation,regulating angiogenesis,assisting protein processing and folding;in addition,researchers have also found that CALR is highly expressed in esophageal cancer,breast cancer,pancreatic cancer and other tumors,and has a significant positive correlation with tumor invasion,metastasis and prognosis.In gastric cancer,a large number of clinical samples,TCGA and oncomine database analysis showed that CALR was highly expressed in gastric cancer,and was related to lymph node metastasis,invasion and prognosis of gastric cancer;however,the specific molecular mechanism of calr regulating gastric cancer metastasis is rarely studied.Therefore,further study on CALR’s role and molecular mechanism in gastric cancer metastasis can provide new ideas for precise treatment of gastric cancer.Objectives1.To investigate the expression and clinical significance of CALR in gastric cancer cells and tissues.2.To investigate the role of CALR in inducing gastric cancer metastasis in vitro and in vivo.3.To elucidate the molecular mechanism of CALR inducing gastric cancer metastasis.Methods1.To investigate the expression and clinical significance of CALR in gastric cancer cells and tissues.1)Search TCGA database and analyze the expression level of CALR in gastric cancer tissues and adjacent tissues,and the correlation between the expression level of CALR in gastric cancer tissues and clinicopathological data of gastric cancer patients.2)Immunohistochemistry and RT-q PCR were adopted to detect the protein and m RNA expression of CALR in gastric cancer cells,gastric cancer tissues and adjacent tissues.3)The correlation between CALR and clinicopathological data was analyzed by clinical follow-up information and oncomine database.Use the Kaplan Meier curve to analyze the correlation between CALR expression and survival prognosis of gastric cancer patients.4)Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of CALR in the serum of gastric cancer patients and normal people.Use ROC curve and AUC area to evaluate the value of using CALR content in serum as a new diagnostic marker.2.To investigate the role of CALR in inducing gastric cancer metastasis in vitro and in vivo.1)Firstly,Shanghai Hanheng Biotechnology Co.,Ltd.constructs the CALR lentivirus knockdown vector and Shanghai Gemma Pharmaceutical Technology Co.,Ltd.constructs the CALR lentivirus overexpression vector.Based on the expression level of CALR in gastric cancer cell lines,MGC-803 and SGC-7901 gastric cancer cell lines were infected respectively,and the stable CALR knockdown MGC-803 cell line and stable CALR overexpression SGC-7901 cell line was screened.2)In vitro Transwell and scratch test were used to detect the effect of overexpression or interference with CALR on the migration ability of gastric cancer cells.3)In vivo,the intraperitoneal dissemination model was used to explore the effect of CALR on the metastatic ability of gastric cancer cells.In the intraperitoneal dissemination model,the experiment was divided into two groups: control group and interference group;each nude mouse in each group was injected with 1 × 10 ^ 6 MGC-803 cells or control gastric cancer cells stably interfering with CALR by intraperitoneal injection Meanwhile,the mice were killed by cervical dislocation,and the abdominal cavity was opened to find the metastatic foci.The number of metastatic foci was counted and photographed.3.To elucidate the molecular mechanism of CALR inducing gastric cancer metastasis.1)Transcriptome sequencing was used to detect differentially expressed molecules and related signaling pathway molecules after stable overexpression of CALR.2)RNA and protein of gastric cancer cell lines stably interfering with or over expressing CALR were collected.RT q PCR and Western blot were used to verify the sequencing results,and further clarify the molecular mechanism of CALR induced gastric cancer metastasis.3)The possible molecular mechanism of CALR inducing gastric cancer metastasis was further explored through MS assays and IP experiments.Results1.Expression and clinical significance of CALR in gastric cancer cells and tissues.1.1 The expression of CALR in gastric cancer cells and tissuesFirst,the gene expression profile analysis of TCGA database showed that the expression level of CALR in gastric cancer tissues was significantly higher than that in adjacent tissues.Next,we further detected the expression of CALR m RNA in 50 pairs of fresh paired gastric cancer tissues and adjacent tissues by RT q PCR.We also detected the expression of CALR protein in 129 pairs of paired gastric cancer tissues and adjacent tissues by immunohistochemical staining.The same conclusion was obtained: the expression of CALR in gastric cancer tissues was significantly increased.In addition,we detected the expression of CALR in gastric cancer cell lines(AGS,BGC-823,MGC-803,SGC-7901,xn0422)and normal gastric mucosal epithelial cells(GES-1),and found that the expression level of CALR in gastric cancer cell lines(AGS,MGC-803,xn0422)was higher than that in normal gastric mucosal epithelial cells(P < 0.05).1.2 The correlation between CALR and clinical markersThrough statistical analysis of clinical follow-up information,clinical correlation analysis showed that: the expression of CALR in gastric cancer tissue was significantly correlated with TNM stage of gastric cancer patients;the expression of CALR in I / II stage gastric cancer group was significantly lower than that in III / IV stage gastric cancer patients(P < 0.05);The expression of CALR in non metastasis group was significantly lower than that in metastasis group(P < 0.05).In addition,the results of immunohistochemistry showed that the expression of CALR was related to the differentiation degree and tumor size of gastric cancer.The expression of CALR was higher in poorly differentiated gastric cancer,and the larger the tumor was,the higher the expression of CALR was(P < 0.05).Through the analysis of oncomine big data(https://www.oncomine.org/),We found that the overexpression of CALR was associated with gastric cancer metastasis and recurrence(P < 0.05),and Kaplan Meier Plotter(http://kmplot.com/)analysis showed that high expression of CALR was associated with poor survival of gastric cancer patients.These results indicate that CALR is up-regulated in gastric cancer,and its up-regulation is significantly correlated with the progression,metastasis,postoperative recurrence and survival prognosis of gastric cancer.The AUC area of serum CALR as a diagnostic marker of gastric cancer was significantly higher than that of traditional markers CA19-9 and carcinoembryonic antigen(CEA)(P < 0.0001).2.To investigate the role of CALR in inducing gastric cancer metastasis in vitro and in vivo.2.1 The effects of CALR on gastric cancer metastasis in vitroIn vitro Transwell and scratch test results showed that interference with CALR expression significantly inhibited the migration of gastric cancer cells AGS(P < 0.05)and MGC-803(P < 0.01).However,overexpression of CALR significantly promoted the migration of SGC-7901 cells(P < 0.05).2.2 The effects of CALR on gastric cancer metastasis in vivoThe results of intraperitoneal dissemination and metastasis model in vivo showed that compared with the mice injected with control MGC-803 cells,the number of visible intraperitoneal nodules in mice injected with MGC-803 cells stably interfering with CALR was significantly reduced(P < 0.01)3.To elucidate the molecular mechanism of CALR inducing gastric cancer metastasis.3.1 Firstly,through the transcriptome sequencing of stable overexpression of CALR cells,we found that overexpression of CALR can significantly increase the expression of SNAI1,ZEB1,ITGB1,FN1,MMP2 and other molecules that play an important role in gastric cancer metastasis;3.2 Through RT-q PCR and WB technology to further verify the sequencing results,we got consistent results: interference or overexpression of CALR can significantly regulate the expression of EMT-related molecular,including SNAI1,ZO-1,E-cadherin,N-cadherin,at m RNA and protein levels.3.3 Through mass spectrometry(MS assays)and immunoprecipitation assay(IP),we detected the relevant proteins that can bind with CALR,and found that CALR can bind with G9 a and Snail to form a complex.3.4 The detection of H3K9 and E-cadherin promoter methylation levels by WB and DNA methylation test showed that in SGC-7901 cell lines with low CALR expression,the DNA methylation levels on H3K9me2 and E-cadherin promoter were lower than those on H3K9me2 and E-cadherin promoter;in AGS and MGC 803 cell lines with high CALR expression,the DNA methylation levels on H3K9me2 and E-cadherin promoter were lower.3.5 Blocking G9 a expression in SGC-7901 cell line overexpressing CALR showed that knockdown of G9 a significantly blocked the inhibition of E-cadherin by overexpressing CALR,and the transwell experiment also showed that knockdown of G9 a significantly inhibited the promotion of overexpressing CALR on gastric cancer cell migration.Conclusions1.This study proved that CALR was highly expressed in gastric cancer tissues and gastric cancer cell lines.The expression level of CALR in gastric cancer is positively correlated with clinicopathological parameters and negatively correlated with patient prognosis.Compared with the traditional diagnostic markers CEA and CA19-9,calr has certain potential research value and can be used as a candidate marker for the diagnosis of gastric cancer.2.In vivo and in vitro experiments confirmed that CALR is an important molecule to promote the invasion and metastasis of gastric cancer cells.3.CALR can induce epithelial mesenchymal transformation of gastric cancer cells,up regulate the expression of cell adhesion related molecules,and induce gastric cancer metastasis.4.CALR can promote the methylation of E-cadherin promoter and H3K9 dimethylation(H3K9me2),inhibit the expression of E-cadherin,and promote the occurrence of EMT and metastasis of gastric cancer by forming a complex with Snail and G9 a. |
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