MiR-30a Targeted Research The Role Of Regulation Beclin1 Protein In Colon Cancer

Objective: Micro RNAshave emerged as importantgene regulators and are recognised as key players in carcinogenesis.Previousstudieshaveshown that miRNA-30 a is a microRNAmolecule that can play a role in tumorsuppression.Researchershave found that the expression of miRNA-30 a was reduced in a variety of malignant tumors,including lung cancer,breast cancer,pancreatic cancer and melanoma and other tumors.It is also confirmed that the decreased expression of miRNA-30 a is often associated with the prognosis of patients,whichhas potential value as the indicator in cancer patients.In thisstudy,To investigate the expression of miRNA-30 a in clinical colon cancersamples and adjacent cancer tissues and to confirm the association between the expression of miRNA-30 a and the clinical indicators of colon cancer patients;2.To investigate the regulation of miRNA-30 a on proliferation,apoptosis,invasion and metastasis and chemosensitivity of colon cancer cells;3.To explore the target proteins of miRNA-30 a in regulating malignant biological behavior of colon cancer cells.Methods: 1)The expression of miRNA-30 a in 50 cases of colon cancer and its adjacent tissues was detected by Realtime PCR,and the correlation between the expression of miRNA-30 a and the clinicopathological features of colon cancer was analyzed.Further analysisshowed that the expression evel of mi R-30 a correlated with the 5-year metastasis-freesurvival rate negatively;2)The lentiviral vector expressing miRNA-30 a was constructed and transfected intohuman colon cancer cell line HCT-116 to enhance the miRNA-30 a expression level.The effects of miRNA-30 a on proliferation,apoptosis,invasion ability and chemosensitivity of colon cancer cells were detected by MTT,flow cytometry and Transwell invasion and migration experiments;3)Bioinformaticssoftware was used to predict andscreen the downstream targetgenes regulated by miRNA-30 a,and then double luciferase reportergene test,realtime PCR and western blot were used to verify the resultsso as to further explore the target of miRNA-30 a regulation;Results: 1)In the clinicalstudy,the expression level of miRNA-30 a in colorectal cancer tissues and adjacent tissues were 5.38 + 1.20 and 1.05 + 0.12,the expression of miRNA-30 a in colorectal cancer wassignificantlyhigher than that in adjacent normal tissues,the difference wasstatisticallysignificant(P<0.05).Further analysis of miRNA-30 a expression level in patients with colon cancersurvival rates,the relationship between the discovery of miRNA-30 ahighgroup expression ofgroup(24 cases)of 5 years without metastasissurvival rate compared to the miRNA-30 a lowgroup expressiongroup(26 cases)significantly increased(P<0.05);2)The median expression of miRNA-30 a was bounded by 50 cases of colorectal cancer patients divided into twogroups: miRNA-30 ahigh expressiongroup and miRNA-30 a low expressiongroup.Correlation analysis of the relationship between the expression level of miRNA-30 a and rectal cancer clinical pathological featuresshowed that no correlation was found between the expression level ofgender,age,degree of differentiation and miRNA-30a;and the expression level of miRNA-30 a wassignificantly correlated with Tstaging,lymph node metastasis the degree of infiltration(P<0.05);3)After miRNA-30 a overexpression lentiviral vectors were constructedsuccessfully,the recombinant lentiviral vector expressing miRNA-30 a wassuccessfully constructed and identified.Thescreening resultsshowed that lentiviral vector transfected with miRNA-30a(LV-hsa-mir-30a)or empty vector NC(LV-hsa-mir-NC)infection inhuman colon cancer cell line HCT-116,visible above about 80% of colon cancer cellsshowedgreen fluorescence,confirmed that the miRNA-30 a lentiviral vector infection;4)Realtime PCR was used to detect the expression of miRNA-30 a in colon cancer cells infected with LV-hsa-mir-30 a or LV-hsa-mir-NC in HCT-116 cells.We found that the expression level of miRNA-30 a was increasedsignificantly by the infection of LV-hsa-mir-30 a,and thestatistical results confirmed that mi R-30 a expression abundance is 1408 times of negative controlgroup cells,the difference wasstatisticallysignificant(P<0.001);5)We then explored the role of miRNA-30 a in regulating the proliferation of colon cancer cells and thesensitivity to oxaliplatin on the basis of overexpression of colon cancer cells.MTT testshowed that overexpression of miRNA-30 asignificantly inhibited compared to the controlgroup(P<0.05).After the addition of oxaliplatin,colon cancer cell proliferation was inhibitedsignificantly,and increased expression of miRNA-30 a also enhanced the thesensitivity of colon cancer cells to oxaliplatin(P<0.05,compared with control cells +oxaliplatin).We investigated the effect of enhanced expression of miRNA-30 a on migration ability of colon cancer cells by Transwell.Thehigh concentration ofserum in the lower chamber of Transwell can induce the migration of colon cancer cells into the lower chamber.The results of cell migration were detected bystaining.Statistical analysisshowed that the number of cells expressed miRNA-30 a in the colon cancer cell line HCT-116,wassignificantly lower than that of the blank control cell(P<0.05).In addition,the rate of migration of miRNA-30 a HCT-116 cells wassignificantly decreased by 43.2%(P<0.05)compared with that in the blank controlgroup after administration of oxaliplatin.Based on the model of HCT116 cells with overexpression of miRNA-30 a,we examined the enhanced expression of miRNA-30 a on apoptosis of HCT116 cell level and chemotherapysensitivity,the results of flow cytometryshowed that the apoptosis rate of HCT116 cells in the controlgroup is 1.80 + 0.17%,the negative controlgroup the apoptosis ratio was 3.63 + 0.14%,the proportion of the apoptosis of HCT116 cells overexpressing miRNA-30 a was 3.86 + 0.09%,there was nosignificant difference.But after the addition of oxaliplatin,the apoptosis rate of miRNA-30 a overexpressing HCT116 cells increased to18.24 + 0.56%,which wassignificantlyhigher than that in the controlgroup(3.84 + 0.03%,P < 0.01);6)Bioinformatics prediction combined with targetgene function analysis,screeningget Beclin1 as miRNA-30 a regulation of the downstream targetgenes.Dual luciferase reportergenesystem detect further confirmed the targetsequence,Realtime PCR and Western blot results confirm miRNA-30 a can inhibit colon cancer Beclin1 protein expression of HCT-116 cells.Conclusion: 1)The expression of and miRNA-30 a in colon cancer tissue was decreasedsignificantly,and is closely related with the prognosis of patients.the abnormal expression of miRNA-30 a was closely related to the malignant phenotype and prognosis of colon cancer patients;2)The up-regulation of miRNA-30 a cansignificantly inhibit the proliferation,invasion and metastasis of colon cancer cells,and promote the apoptosis of colon cancer cells and enhance thesensitivity of chemotherapy;3)Beclin1 are downstream targets of miRNA-30 a regulation,and miRNA-30a-Beclin1 may become targets of colon cancer treatment.