| Gastric cancer is one of the most common cancers worldwide.Its incidence rate ranks fifth in cancer,and its mortality rate ranks third.Incidence rate is the highest in East Asia.Gastric cancer has high frequency of scnv and GIS,and it is easy to have peritoneal metastasis,which makes it difficult to accurately evaluate the therapeutic effect of drug therapy by existing imaging evaluation standards.Although in recent years,with the treatment of drugs,the average five-year survival rate of gastric cancer patients has improved,but relatively speaking,the prognosis of gastric cancer patients is still very poor.Therefore,it is very important and urgent to find a target for the treatment of gastric cancer.Ubiquitination is the key to maintain normal cell life.In recent years,many E3 ubiquitin ligases have been widely reported as potential targets of tumor therapy.HECTD3 is a member of hect E3 ubiquitin ligase family.It is reported that HECTD3 is highly expressed in breast cancer,ovarian cancer and human esophageal squamous cell carcinoma,and participates in apoptosis and immune regulation,which promotes the proliferation and metastasis of cancer cells.However,the role and mechanism of HECTD3 in other tumors are not fully understood,especially in gastric cancer.So this paper aims to explore the mechanism of HECTD3 in gastric cancer.1.HECTD3 is highly expressed in gastric cancer and the prognosis of gastric cancer patients was poor.To explore the role of HECTD3 in gastric cancer,we first analyzed the prognosis of HECTD3 in gastric cancer patients through the gastric cancer database.The results showed that HECTD3 was highly expressed and the prognosis of gastric cancer patients was poor.In order to further explore the expression of HECTD3,we used western The protein and m RNA expression of HECTD3 in normal gastric adenocarcinoma cells GES-1 and gastric cancer cells SGC7901,MKN45,HGC27,MGC803 were detected by Western blot and q RT-PCR.The results showed that HECTD3 was highly expressed in gastric cancer cells.These results showed that hectd3 played a role of oncogene in gastric cancer,which may be a promising target and marker for the treatment of gastric cancer patients.2.HECTD3 regulates proliferation and cell cycle of gastric cancer cellsIn order to investigate the effect of HECTD3 on the proliferation and cell cycle of gastric cancer cells,we constructed the interference plasmids shHECTD3 and overexpression HECTD3.In view of SGC7901 and MKN45,we downregulated the expression of HECTD3,and detected the effect of down regulated HECTD3 on the proliferation of gastric cancer cells by MTT and BrdU experiments.The results showed that down regulated HECTD3 could inhibit the proliferation of gastric cancer cells.In order to further explore,we used flow cytometry and Western blot to detect the effect of down-regulation of HECTD3 on cell cycle arrest and expression of related cyclins in gastric cancer cells.The results showed that after down regulating the expression of HECTD3,the proliferation of gastric cancer cells was significantly inhibited,the cell cycle was blocked in G1/S phase,and the expression of related cyclin CDK2 and CDK4 was down regulated.After that,in order to verify that the effect of down-regulation of HECTD3 on the proliferation of gastric cancer cells is not caused by the Miss target effect,we carried out a recovery experiment on the gastric cancer cells down-regulated HECTD3,and detected the effect of recovery of HECTD3 on the proliferation and cycle arrest of gastric cancer cells through MTT experiment,BrdU experiment and flow cytometry.The results showed that after the recovery of HECTD3,the inhibition of cell proliferation was saved and cell cycle arrest was relieved.These results indicate that HECTD3 plays a role as a oncogene in gastric cancer,which provides a theoretical basis for the treatment of gastric cancer.3.HECTD3 inhibits apoptosis of gastric cancer cellsTo explore the influence of HECTD3 on gastric cancer cell migration and apoptosis,we use the Transwell experiment to test the influence of down-regulation HECTD3 on gastric cancer cell migration ability,the results show that down-regulation HECTD3 can inhibit gastric cancer cells’ ability to migrate.Then we used flow cytometry and Western blot to detect the effect of down-regulation of HECTD3 on apoptosis and expression of apoptosis related proteins.The results showed that after down regulating HECTD3 in SGC7901 and MKN45,the apoptosis of gastric cancer cells was significant.The expression of apoptotic protein was consistent with that of flow cytometry,and the expression of c-PARP and P53 was up-regulated.In gastric cancer cells,HECTD3 was down regulated to restore the cells,flow cytometry analysis showed that apoptosis decreased,Western blot test detected the down regulated expression of c-PARP.These results indicate that HECTD3 plays a role in promoting cancer gene by inhibiting apoptosis in gastric cancer.4.HECTD3 promote the ability in vitro cloning of gastric cancer cells and tumor growth in miceBased on the above results,we also explored the effect of HECTD3 on the ability of gastric cancer cell clone formation in vitro and tumor formation in vivo.Soft Arga and subcutaneous tumorigenesis were used to detect the effect of down-regulation of HECTD3 on the ability of clone formation in vitro and in vivo.The results of soft Arga showed that after down-regulation of HECTD3,the clone number of gastric cancer cells decreased significantly,while after down-regulation of HECTD3,the clone number in vitro recovered.Similarly,the subcutaneous tumorigenesis of mice showed that the tumor weight and size of mice after down-regulation of HECTD3 were significantly lower than those of the control group,and the tumor volume and weight were recovered after rescuing the expression of HECTD3.At last,the expression of HECTD3 and proliferation marker Ki67 were detected by immunohistochemistry.The results of immunohistochemistry showed that the expression of Ki67 and HECTD3 in the experimental group was lower than that in the control group,and the expression of Ki67 and HECTD3 recovered after the recovery of HECTD3 expression.The above results showed that HECTD3 could promote the ability of gastric cancer cells to clone in vitro and tumorigenesis in vivo.In conclusion,we first explored the mechanism of HECTD3 in gastric cancer,HECTD3 high expression in gastric cancer cells,and is related to the prognosis of patients with gastric cancer.HECTD3 can regulate the cell cycle,promote gastric cancer cell proliferation.In addition,HECTD3 can significantly inhibit the apoptosis of gastric cancer cells,and promote gastric cancer cells in vitro and in vivo tumor ability.This will provide a theoretical basis for the treatment and clinical research of gastric cancer patients,and HECTD3 may become an important target of gastric cancer detection and treatment. |
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