| Part I The expression of lnc RNA AC093818.1 in gastric cancer tissue and its relationship with gastric cancer progressionGastric cancer is one of the most common malignant tumors.It is very prone to lymphatic metastasis and blood metastasis.Distant metastasis is the main cause of death in patients with gastric cancer.However,the molecular mechanism of invasion and metastasis of gastric cancer is still not clear.Lnc RNA(long non-coding RNA)is a class of non coded RNA molecules with length larger than 200 nt and does not have open reading frames.It plays an important role in many biological processes,such as immune response,pluripotent stem cell pluriability and cell proliferation and apoptosis differentiation.AC093818.1,a natural lnc RNA in human body,is located at 173420153-173421135 of chromosome 2,and is found to be highly expressed in tumor tissues of highly metastatic gastric cancer patients,up to 5 times more.Scholars have not systematically studied the role of AC093818.1 in the invasion and metastasis of gastric cancer and its related regulatory mechanisms.Objective: To explore the correlation between the expression level of AC093818.1 in gastric cancer tissue and tumor invasion and metastasis.Method: From 2016 to 2017,20 cases of gastric cancer diagnosed as gastric cancer were collected from the Cancer Center of Guangzhou Medical University,including 10 cases of lymph node metastasis,and 10 cases of metastasis were not found.Through literature analysis,5 abnormal high expression lnc RNA(AC093818.1,CTD-2541M15.1,BC047644,RP11-597M12.1,RP11-40A13.1)in high metastatic gastric cancer were obtained to verify the expression of the 5 lnc RNA in gastric cancer tissues with metastasis and non-metastasis,and the characteristics of the presence of the adjacent beds were analyzed and the expression of lnc RNA was studied.The relationship with clinical features.The most suitable lnc RNA for the study was screened out and analyzed for its expression in gastric cancer tissues with metastasis and non-metastasis.The relationship between tumor size,clinicopathological stage,invasion degree and lymph node metastasis was also analyzed.Result: In 5 lnc RNA(AC093818.1,CTD-2541M15.1,BC047644,RP11-597M12.1,RP11-40A13.1)in high metastatic gastric cancer,the high expression of AC093818.1 in high metastatic gastric carcinoma was very significant(p<0.01),but the expression of other 4 lnc RNA in metastatic and non metastatic gastric cancer tissues was not significantly different..Conclusion: The clinical feature analysis showed that the expression level of AC093818.1 was associated with the clinical stage and lymph node metastasis and distant metastasis of gastric cancer(p<0.05,p<0.01,p<0.05).Part II The effect of AC093818.1 on invasion and metastasis of gastric cancer cells through PDK1Background and Objective: The main biological characteristics of malignant tumor are invasion and metastasis,and the invasion and metastasis process of the tumor is very complex,which has a great influence on the clinicopathological features and prognosis of the patients.The expression of AC093818.1 in tumor tissues of gastric cancer patients was significantly correlated with tumor size,clinicopathological stage,invasion degree and lymph node metastasis.PDK1(3-phosphoric acid inositol dependent protein kinase 1)is a protein kinase that mainly regulates the activation of the AGC protein kinase family,which plays a very important role in many signal pathways.However,the expression of AC093818.1 in gastric cancer cells and its relationship with PDK1 have not been verified.In this study,we screened the gastric cancer cell line with the highest AC093818.1 expression and gastric cancer cell line with the lowest AC093818.1 expression,and observed the relationship between AC093818.1 and PDK1 and the influence on the invasion and metastasis of gastric cancer cells.Methods: 1.cell screening: in BGC82,MGC803,MKN28,MKN45,SGC7901 five cell lines,screening AC093818.1 and PDK1 expression relatively high and low cell lines.2.cell line construction: AC093818.1 overexpressed and inhibited the expression of virus.Through resistance screening,the MKN28-AC093818.1 cell line expressing AC093818.1 and an empty virus control MKN28-CTR cell line were constructed,and the MGC803-si AC093818.1 cell line that inhibited the expression of AC093818.1 and an empty virus control MGC803-CTR cell line were constructed.3.Transwell assay was used to detect cell migration and cell invasion in MKN28-CTR?MKN28-AC093818.1?MGC803-CTR and MGC803-si AC093818.1.4.cell scratch test was used to detect cell migration in MKN28-CTR ?MKN28-AC093818.1?MGC803-CTR and MGC803-si AC093818.1.5.The expression of PDK1,PDK1,MMP2,MMP9,E-cadherin and Vimentin in the cells of MKN28-CTR?MKN28-AC093818.1?MGC803-CTR and MGC803-si AC093818.1 were detected by RT-PCR.6.The expression of PDK1 and MMP2,MMP9,E-cadherin and Vimentin in MKN28-CTR?MKN28-AC093818.1?MGC803-CTR and MGC803-si AC093818.1 were detected by Western blot..Result: Compared with MKN28-CTR,the expression of PDK1 factor in gastric cancer cells in MKN28-AC093818.1 was up-regulated,the expression of invasion and metastasis related protein MMP2,MMP9 and Vimentin was also up-regulated,and the expression of E-cadherin was down regulated,and the invasion ability of gastric cancer cells in MKN28-AC093818.1 increased,and the difference was statistically significant(P<0.05);the MKN28-AC093818.1 of gastric cancer cells The migration ability increased,the difference was statistically significant(P<0.05).Compared with the MGC803-CTR the host gene of the gastric cancer cells in the MGC803-si AC093818.1 was PDK1,the invasion and metastasis related factors MMP2,MMP9 and Vimentin were down regulated,the expression of E-cadherin was up regulated,the invasive ability of gastric cancer cells in the MGC803-si AC093818.1 decreased,and the difference was statistically significant(P<0.05).The migration ability of cells decreased,and the difference was statistically significant(P<0.05).Conclusion: The increase of AC093818.1 expression level can significantly enhance the invasion and migration ability of MKN28 gastric cancer cells.Overexpression of AC093818.1 promotes the invasion and metastasis of gastric cancer cells by upregulating PDK1 factor and indirectly regulating the expression of invasion and metastasis genes.Inhibition of AC093818.1 significantly inhibited invasion and migration of MGC803 gastric cancer cells.Inhibition of AC093818.1 inhibits the invasion and metastasis of gastric cancer cells by inhibiting PDK1 factor and indirectly regulating the expression of invasion and metastasis genes.Part III The effect of AC093818.1 inhibition on nude mice model of gastric cancer metastasisBackground and Objective: It has been found that in gastric cancer,the expression of PDK1 m RNA and protein is higher in tumors compared with adjacent normal tissues.In addition,compared with low expression of PDK1,patients with high PDK1 expression survived for a short time.Therefore,the expression of PDK1 can be used as a prognostic biomarker of [8] for this kind of cancer.Other studies have shown that inhibition of PDK1 can inhibit invasion and metastasis of cancer through multiple pathways.PDK1 is a potential target for cancer therapy.In the first two parts of the experiment,the expression of AC093818.1 in gastric cancer tissue and gastric cancer cells is positively correlated with the level of PDK1 m RNA,and AC093818.1 through PDK1 promotes the invasion and metastasis of gastric cancer cells.No suitable PDK1 inhibitors have been reported yet,and the reduction of PDK1 by inhibiting the expression of AC093818.1 has not been studied.In this part,we will explore the effect of down regulated AC093818.1 on the proliferation and invasion and metastasis of gastric cancer cells,and the correlation with invasion and metastasis of gastric cancer by constructing a stable cell line of AC093818.1 inhibition expression.Methods: 1.construction of stable cell lines: using the AC093818.1 constructed in the second part of the experiment to inhibit the expression of the virus,and to construct a MKN28 AC093818.1 inhibiting expression of the stable cell line MKN28-si AC093818.1 and the control cell line MKN28-CTR.2.animal model experiment: a sufficient amount of MKN28-CTR,MKN28-si AC093818.1,MGC803-CTR and MGC803-si AC093818.1 cell lines were cultured.The above 4 groups of gastric cancer cells were injected into the tail vein of BALB/C nude mice to construct the metastatic tumor model,which were MKN28-CTR,MKN28-si AC093818.1,MGC803-CTR and MGC803-si AC093818.1.3.tumor tissue sections were prepared,and necrosis of tumor tissues was observed by HE.4.The m RNA expression level of MMP2,MMP9,E-cadherin and Vimentin in MKN28-CTR,MKN28-si AC093818.1,MGC803-CTR and MGC803-si AC093818.1 was detected by RT-PCR.5.The protein level of MMP2,MMP9,E-cadherin and Vimentin in MKN28-CTR,MKN28-si AC093818.1,MGC803-CTR and MGC803-si AC093818.1 was detected by western blot.Result: In the animal experiment,compared with the MKN28 control group,there was no significant difference between the tumor size and the tumor size in the MKN28 inhibition expression group(P>0.05).There was no significant difference in the degree of tumor necrosis in HE,and there was no significant difference in the expression of MMP2,MMP9,E-cadherin and Vimentin(P>0.05),and the metastasis of the tumor with MGC803 control group.Compared with nude mice,the tumor occurrence date of MGC803 inhibited expression group was late,and the tumor size decreased significantly(P<0.05).HE found that the degree of tumor necrosis was obviously elevated,the expression of MMP2,MMP9 and Vimentin were down regulated,and the expression of E-cadherin was up-regulated..Conclusion: In the tumors which has high expression of AC093818.1,the inhibition of AC093818.1 expression can be mediated by down regulation of its host gene PDK1 and indirectly regulating the expression of related invasion and metastasis genes,thus promoting tumor necrosis and reducing tumor invasion and metastasis.In the tumors which has lower expression of AC093818.1,inhibition of AC093818.1 expression had no significant effect on tumor necrosis and invasion and metastasis. |
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