| Background:Cervical cancer is a kind of tumor of the female reproductive system and one of the most common female malignant tumors.In recent years,its morbidity and mortality have gradually increased,seriously threatening women's health.At present,the mechanism of cervical cancer development has not been fully elucidated.In recent years,studies have found that long non-coding RNA(lnc RNA)plays a key role in the development of cervical cancer.Some studies have shown that lnc RNA can be used as a competitive endogenous RNA(ce RNA),competitively combined with micro RNA(mi RNA)to inhibit its activity and function,thereby regulating the expression of downstream target gene m RNA,and participate in tumor development.Lnc RNA TDRG1 is a long non-coding RNA discovered in recent years and can regulate gene expression.newly discovered long-chain non-coding RNA that can regulate gene expression in recent years.It has been reported in the literature that it is related to tumor malignant progress in testicular germ cell tumors,endometrial cancer,and ovarian cancer.In order to regulate the related factors of reproductive system tumors,however,the regulatory mechanism of TDRG1 in the progression of cervical cancer is still unclear,and we need to further explore.Objective:To explore the mechanism of lnc RNA TDRG1 on the cell proliferation,migration and invasion in cervical cancer cells.Part1 The expression and clinical significance of TDRG1 in cervical cancer tissueMethods:1.Collect 30 cases of cervical cancer tissues and 30 cases of normal cervical tissues,and use q RT-PCR to detect the expression level of TDRG1;2.Analyze the relationship between the expression level of TDRG1 and the clinicopathological characteristics and prognosis of cervical cancer patients;3.The TDRG1 target mi R-326 was analyzed by bioinformatics software,q RT-PCR was used to detect the expression of mi R-326 in cervical cancer tissue,Spearman correlation analysis was used to analyze the correlation between mi R-326 expression and TDRG1 expression;4.The downstream target MAPK1 of mi R-326 was analyzed by bioinformatics software.The expression of MAPK1 m RNA in cervical cancer tissue was detected by q RT-PCR.Spearman correlation analysis was used to analyze the correlation between the expression of MAPK1 and the expression of TDRG1 and mi R-326.Results:1.The expression level of TDRG1 in cervical cancer tissue is higher than that in normal cervical tissue;2.TDRG1 expression level was significantly positively correlated with FIGO stage(IIb-IIIa),tumor differentiation(low),cervical infiltration depth(?2 / 3),and lymph node metastasis(yes)in patients with cervical cancer;3.The average survival time of TDRG1 high expression in cervical cancer patients is less than that of TDRG1 low expression;4.The results of q RT-PCR showed that the expression level of mi R-326 in cervical cancer tissue was lower than that in normal cervical tissue.Spearman correlation analysis showed that the expression of TDRG1 and mi R-326 were negatively correlated;5.The expression of MAPK1 m RNA in cervical cancer tissue was higher than that in normal cervical tissue.Spearman correlation analysis showed that MAPK1 expression was positively correlated with TDRG1 expression and negatively correlated with mi R-326 expression.Conclusions:1.TDRG1 is highly expressed in cervical cancer tissues;2.The high expression of DRG1 enhances cervical cancer invasion and is positively correlated with FIGO stage(IIb-IIIa),degree of differentiation(low),lymph node metastasis(yes),cervical infiltration depth(?2 / 3),and poor prognosis in cervical cancer patients;3.The expression level of TDRG1 was negatively correlated with the expression of mi R-326;4.The expression level of TDRG1 was negatively correlated with MAPK1 expression.Part2 The effect of TDRG1 on the biological behavior of cervical cancer cellsMethods:1.Use q RT-PCR to detect the expression level of TDRG1 in human cervical immortalized squamous cells(Ect1 / E6E7)and human cervical cancer cell(Hela,SIHA,Ca Ski),and select the target research object;2.The expression of TDRG1 was down-regulated in Hela cells,and the biological behavior of Hela cells was detected by colony forming assay,MTT assay,flow cytometry assay,wound healing assay,and transwell assay with Matrigel.Results:1.The expression level of TDRG1 in Hela,SIHA,and Ca Ski cells was higher than that of Ect1 / E6E7.Because the expression level of TDRG1 in Hela cells was higher,it was selected for subsequent experiments;2.The results of colony forming assay showed that the number of cell clones that down-regulated TDRG1 expression decreased;MTT assay results showed that the cells that down-regulated TDRG1 expression decreased in absorbance at 490 nm;the results of flow cytometry assay showed that down-regulated TDRG1 expression caused cell cycle arrest at G0 / G1 phase,the detection of apoptosis results showed that down-regulation of TDRG1 expression increased apoptosis;3.The results of wound healing assay showed that cells with down-regulated TDRG1 expression healed more slowly;the results of transwell assay showed that the number of cells with down-regulated TDRG1 expression decreased.Conclusions:1.TDRG1 is highly expressed in human cervical cancer cell lines;2.Down-regulating the expression of TDRG1 inhibits the proliferation,migration and invasion of cervical cancer cells.Part3 TDRG1 promotes cell proliferation,migration and invasion via the axis of mi R-326 / MAPK1 in cervical cancerMethods:1.Using dual luciferase reporter assay and RIP to verify the relationship between TDRG1 and mi R-326;2.Co-transfect Hela cells with TDRG1 si RNA and mi R-326 inhibitor,and use colony forming assay,MTT assay,flow cytometry assay,wound healing assay,and transwell assay with Matrigel to study the changes of biological behavior of cervical cancer cells;3.q RT-PCR and western blot were used to detect the changes in m RNA and protein expression levels of target gene MAPK1 up-regulated / down-regulated by TDRG1 or mi R-326.Results:1.The dual luciferase reporter assay shows that mi R-326 can reduce the luciferase activity of TDRG1,and the RIP shows that TDRG1 can specifically enrich mi R-326;2.The results of colony forming assay showed that the number of cell clones that down-regulated the expression of TDRG1 and mi R-326 increased;the results of MTT assay showed that the absorbance of cells that down-regulated the expression of TDRG1 and mi R-326 increased at 490 nm;flow cytometry assay detection of cell cycle results showed that the down-regulation of TDRG1 and mi R-326 expression had a weaker blocking effect on G0 / G1 phase,and the detection of apoptosis showed that the apoptosis of TDRG1 and mi R-326 was reduced;3.The results of wound healing assay showed that the healing distance of cells with down-regulated expression of TDRG1 and mi R-326 increased;the results of transwell assay showed that the number of cell invasion with down-regulated expression of TDRG1 and mi R-326 increased;4.MAPK1 m RNA and protein expression was negatively correlated with mi R-326expression;MAPK1 m RNA and protein expression was positively correlated with TDRG1 expression.Conclusions:1.TDRG1 can specifically bind to mi R-326 and their expression levels are negatively correlated;2.Down-regulation of mi R-326 expression can partially reverse the inhibitory effect of down-regulation of TDRG1 expression on cervical cancer cell proliferation,migration and invasion;3.mi R-326 inhibits MAPK1 expression,TDRG1 enhances MAPK1 expression;TDRG1 can be used as a ce RNA to competitively adsorb to mi R-326,inhibit mi R-326 activity and function,and upregulate MAPK1 expression levels;4.TDRG1 regulates the proliferation,migration and invasion of cervical cancer cells by regulating the mi R-326 / MAPK1 axis to exert its carcinogenicity and promote the occurrence and development of cervical cancer;5.TDRG1 can be used as a potential target to provide new ideas and new directions for the diagnosis,treatment and prognostic evaluation of cervical cancer. |
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