| Background:The incidence and death rate of gastric cancer have remained high,seriously affecting people's health.Of all malignancies,gastric cancer is the fourth most common malignancy,and in the world,mortality caused by gastric cancer ranks second in all cancer mortality rates.There are no obvious symptoms in the early stage.A large part of the patients was diagnosed at the middle or the late stages of cancer,and the prognosis is often poor.In the diagnosis and treatment of gastric cancer,the early diagnosis rate of gastric cancer is low,the diagnostic effect is poor,the accuracy of diagnosis needs to be further improved,and chemotherapy is less effective in the therapy of gastric cancer,and it is easily tolerated,and relapse is caused by improper treatment.The prognosis of stomach cancer patients is not good.Therefore,there is an urgent need for biomarkers that can accurately diagnose gastric cancer early and therapeutic targets that can be applied to clinical treatment.MicroRNA,or miRNA,is a small RNA in non-coding RNAs.It is about 22 nucleotides in length and is involved in post-transcriptional regulation in animals and plants.MiRNAs bind to specific target sites of target genes,leading to degradation of target gene mRNA or inhibition of target gene translation.MiRNA regulates the expression of a variety of tumor-associated genes in cells and plays an important role in regulating the cell cycle,apoptosis,cell differentiation,tissue inflammatory response,and tumor invasion and metastasis.The occurrence and development of tumors are accompanied by the abnormal expression of certainly specific miRNAs.The types of miRNAs in different tumor tissues are also different.Even if they are the same tumor,the types and expression of miRNAs at different stages of disease are not the same.Therefore,based on the tissue specificity and temporal specificity of miRNAs,miRNAs are expected to be used as molecular markers for the early diagnosis and disease detection of various malignant tumors,and miRNAs have the function of regulating gene expression and regulating many physiological functions of cells,and many studies have also been conducted.It is pointed out that miRNA can be used as an important therapy to treat malignant tumors.Several studies have reported that miR-135b-5p plays an important role in many malignant tumors(such as non-small cell lung cancer and breast cancer).MiR-135b-5p participate in the regulation of the apoptosis of cancer cells and the tolerance of chemotherapeutic drugs in colon cancer by inhibiting the expression of TGFBR2 and LAST2,and miR-135b-5p can inhibit the expression of EVI5 and RECK and regulate the metastasis of hepatoma cells.However,the specific expression level and function of miR-135b-5p in gastric cancer and potential mechanism have not been studied.Through using miRNA database,researchers can quickly and effectively explore potential target genes for miRNAs.We have discovered that CMTM3 may be a target gene for miR-135b-5p and are involved in the regulation of tumor cell function.The CMTM family is also known as the chemokine-like factor family(CKLFS).Several studies have pointed out that CMTM3,a tumor suppressor gene,plays an important role in inhibiting tumor development and progression in a variety of malignant tumors(such as prostate cancer and renal clear cell carcinoma)in the section of tumor metastasis.CMTM3 has also been related to the metastasis of gastric cancer in recent studies,but the specific regulation mechanism of CMTM3 in gastric cancer is still not clear.This study aimed to explore how miR-135b-5p regulates the occurrence and development of gastric cancer and its underlying mechanism.In summary,the expression levels and potential functions of miR-135b-5p in gastric cancer tissues and gastric cancer cells are explored.The targeted gene of miR-135b-5p is predicted by database and the expression of target genes in gastric cancer is further explored.The level and related functions as well as the correlation with miR-135b-5p expression provide an important theoretical basis for miR-135b-5p as a molecular marker for early diagnosis,disease monitoring and tumor treatment in gastric cancer.Aim:1.To detect expression levels of miR-135b-5p in gastric cancer tissues,paracancerous tissues,and gastric cancer cell lines.2.To explore the specific role of miR-135b-5p in gastric cancer cells.3.To predict and identify miR-135b-5p specific potential target genes and examine whether they are associated with miR-135b-5p function.4.To detect target gene's expression in gastric cancer tissues,paracancerous tissues,and gastric cancer cell lines.5.To explore the function of target genes in gastric cancer cells.6.To explore the effect of miR-135b-5p knockdown on tumors in animal models.Methods:1.The expression level of miR-135b-5p in cancer tissue samples and pericancerous tissue samples from 20 patients diagnosed as gastric cancer by pathological diagnosis was detected by fluorescent real-time quantitative PCR.And the expression level of miR-135b-5p was detected in four gastric cancer cell lines(HGC-27,SGC-7901,BGC823 and MKN45)and a human-derived normal gastric epithelial cell line(GES-1)through using fluorescent real-time quantitative PCR.2.Firstly,we select a suitable cell line to construct miR-135b-5p knockdown gastric cancer cell lines.Cell viability of miR-135b-5p knockdown gastric cancer cells was detected by CCK8 experiment.Changes in the proportion of each cell cycle was detected by flow cytometry,cell apoptosis was detected by flow cytometry,and cell invasion was detected by transwell assays.3.Using three classical miRNA databases(TargetScan,miRDB and miRanda)to perform miR-135b-5p target gene prediction,and then predicting the potential target gene CMTM3 of miR-135b-5p,using fluorescence quantitative PCR to verify the correlation between the expression of CMTM3 and the expression of miR-135b-5p.The CMTM3-3'-UTR WT and CMTM3-3'-UTR Mut plasmids were designed and constructed and transferred into gastric cancer cells,and luciferase assay was used to verify whether CMTM3-3'-UTR is the target site of miR-135b-5p.The protein expression level of CMTM3 was measured after knockdown of miR-135b-5p by Western blotting.4.Fluorescent real-time quantitative PCR was used to detect the expression level of miR-135b-5p target gene CMTM3 in cancer tissues and pericancerous tissues of gastric cancer patients,and in four gastric cancer cell lines(HGC-27,SGC-7901,BGC823 and MKN45)and a normal gastric epithelial cell line(GES-1).5.Select appropriate cell lines to construct CMTM3 overexpressing gastric cancer cell line.We use CCK8 experiment to detect the effect of CMTM3 overexpression on cell viability of gastric cancer cells,and use flow cytometry to detect the cell cycle and apoptosis after CMTM3 overexpression.Transwell assay was used to detect the change of tumor cell invasive ability after overexpression of CMTM3.6.A nude mouse subcutaneous tumor model was constructed using a knockdown miR-135b-5p gastric cancer cell line and a negative control gastric cancer cell line.The volume of subcutaneous tumors and mouse body weight were continuously detected.Subcutaneous tumors were harvested after sacrifice and the expression of miR-135b-5p and CMTM3 proteins were detected by fluorescent quantitative PCR.Results:1.Detection of miR-135b-5p expression level in gastric cancer tissues,adjacent tissues and gastric cancer cell linesThe expression level of miR-135b-5p in cancer tissue of patients with gastric cancer was significantly higher than that in the normal control group(p<0.01).The expression level of miR-135b-5p in gastric cancer cell lines was significantly higher than that in normal gastric epithelial cell lines(p<0.001),and miR-135b-5p has the highest level of expression in the gastric cancer cell line SGC-7901.2.Detecting the change of cell proliferation ability and invasive ability of gastric cancer cells after miR-135b-5p knockdownIn the CCK8 experiment,cell viability in the knockdown miR-135b-5p group was significantly lower than that of the untreated or control group,and the difference was statistically significant(p<0.01).Flow cytometry experiments showed that compared with untreated group and normal group,knockdown of miR-135b-5p increased the proportion of G1 cells and inhibited Gl/S phase transition;knocking down miR-135b-5p increased early apoptosis and late apoptosis cells and promoted apoptosis.And in transwell experiments,knocking down miR-135b-5p can attenuate the invasive ability of gastric cancer cells.3.Prediction and identification of target gene CMTM3 of miR-135b-5pThree miRNA database predictions show that CMTM3 is a target gene for miR-135b-5p,and luciferase assay results confirm that the CMTM3-3'-UTR region is a target site for miR-135b-5p.qRT-PCR experiments showed that the expression level of miR-135b-5p was negatively correlated with the expression level of CMTM3.We detected that protein levels of CMTM3 decreased significantly after knocking down miR-135b-5p using WB technology.4.Detection of CMTM3 expression in gastric cancer tissues,adjacent tissues and gastric cancer cell linesReal-time fluorescence quantitative PCR assays showed that the expression of CMTM3 in cancer tissue was significantly lower than that in the normal control group.Moreover,the expression level of CMTM3 in gastric cancer cell lines was significantly lower than that in normal gastric epithelial cell lines(p<0.001),and the expression level of CMTM3 is the lowest in gastric cancer cell line SGC-7901.5.Detecting changes of cell proliferation and invasive ability of gastric cancer cells by CMTM3 over-expressionCCK8 experiments,flow cytometry,and transwell invasion experiments showed that the cell viability of the overexpressed CMTM3 group was significantly lower than that of the untreated group or the control group,and the difference was statistically significant(p<0.01).In untreated and normal groups,overexpression of CMTM3 increased the proportion of cells in G1 phase and inhibited G1/S phase transition;overexpression of CMTM3 apoptotic cells increased significantly,overexpression of CMTM3 promoted apoptosis;overexpression of CMTM3 attenuated the invasive ability of gastric cancer cells.6.Construction of nude mice tumorigenesis model to detect the effect of miR-135b-5p on tumorigenicity of gastric cancer cellsThe results of subcutaneous tumorigenesis in nude mice showed that subcutaneous tumor volume and body weight in the miR-135b-5p knockdown experimental group were smaller than those in the control group and the untreated group.The results of real-time fluorescence quantitative PCR on harvested subcutaneous tumors showed that miR-135b-5p levels in subcutaneous tumors in the miR-135b-5p knockdown experimental group were lower than those in the control groups(p<0.01).The IHC results showed that knockdown of miR-135b-5p experimental group CMTM3 expression levels were significantly higher than the control groups insubcutaneous tumor tissues.Conclusion:MiR-135b-5p has a high expression level in gastric cancer tissues,and knockdown of miR-135b-5p can significantly reduce the cell proliferation and cell invasion ability of gastric cancer cells.While CMTM3 is a regulatory downstream gene of miR-135b-5p,CMTM3 has a significantly low expression level in cancer tissues,and it can also affect the cell proliferation ability of gastric cancer cells.MiR-135b-5p can regulate the expression of CMTM3 and reduce the tumorigenicity of gastric cancer cells in vivo.MiR-135b-5p is promising as a novel biomarker for clinical diagnosis and targeted therapy of gastric cancer. |
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