Screening For Sex Hormone Dependence Of Female Patients With Non-small Lung Cancer And Its Significance

Non-small lung cancer(NSCLC)has high morbidity and mortality in both male and female tumors.In recent years,the incidence of lung cancer in man has reached a plateau,while the incidence of lung cancer in woman has increased year by year.National Cancer Registry data 2014 show that an estimated 189600 case in female lung cancer was found in China in 2009,accounting for 14.75% of new cases of malignnant tumors,making it second female malignant tumors.The female lung cancer has several different characteristics with the age of the onset of the young tendency,high morbidity and higher risk in never smokers.Although the incidence of the female lung cancer increased partly attributed to the increased number of female smokers,women cooking or passive smoking,women’s susceptibility to smoke,human papillomavirus(HPV)infection or the life pressure of modern society.But some research can not get this conclusion.It can not be ignored that the effect of smoke contributes to squamous lung cell carcinoma.It is also difficult to explain the increase in female lung cancer number and the younger age of female patients with adenocarcinoma.Gender differences affect the different characteristics,disease progression and molecular biology,It determine the therapeutic effect and prognosis of non-small cell lung cancer.s The biggest difference between men and women is different sexal hormones.The most important sexal hormone is estrogen.Estrogen is a steroid hormone showed the highest activity,which closely related with estrogen receptor in high degree,includes estradiol,estriol and estrone.It was mainly secret by the ovarian follicle.Placenta also produced a little in pregnancy.In postmenopausal women,theovaries no longer produce estrogen,and serum estradiol levels were significantly decreased.Studies have shown that estrogen can induce the proliferation of tumor cells in vivo or in vitro,and the anti-estrogen drugs can block this effect.Another seaxal hormone is progesterone,progesterone is also female steroid hormone produc-ed by the ovarian corpus.Luteinizing hormone(LH)is a glycoprotein hormone secret ed by basophilic cells in the anterior pituitary,mainly to promote ovarian ovulation.In the synergistic effect of FSH,the formation of luteal phase and progester-one secretion go on.Follicle-stimulating hormone(FSH)is a glycoprotein hormone secreted by basophilic cells in the anterior pituitary.Its main function is to promote ovarian follicle development and maturation.FSH is secreted by the ovary of the corpus luteum,the main function of it is to promote the endometrium from prolifer-ative to secretory phase.Estrogen receptor(ER)and progesterone receptor(PR)were associated with breast cancer and endometrial cancer,estrogen and progesterone receptor is an important factors for judging the prognosisr and hormone treatment of breast cancer and endometrial cancer.Detection of estrogen receptor and progesterone receptor in endocrine therapy for breast cancer has been widely used in clinical practice and has achieved remarkable curative effect.Thus,The breast cancer and other dependent hormone target tumor is called sexal hormone dependent tumor.Not only that,It was reported there is sex hormone receptors in non target organs of sex hormone in tumor tissue.Previous studies have suggest that sex hormone receptors existed in patients with normal human tissue and the tissue of lung cancer,the level of sex hormone receptors in lung cancer tissues was significantly higher than that of normal people,the lung cancer is also the sex hormone dependent tumor.There are two subtypes of estrogen receptor,estrogen receptor alpha(ERα)and estrogen receptor beta(ERβ).In previous studies,the expression of ERα in lung cancer is still controversial and the expression of ERα in lung cancer is not obvious,lung cancer is mainly expressed in ERβ.ERβ plays an important role in the lung cancer,especially in sex hormone dependent tumors.The mechanism of estrogen and its receptor may be binding to the tumor cytoplasm after the combination of specificreceptors,formating hormone receptor complex.Hormone receptor complexes can pass through the nuclear envelope into the nucleus.Nuclear receptor was bind and form the dimerization monomer,which affact the transcription of DNA(DNA)in the nucleus,a new protein was synthesized and a new tumor was formedAlthough the anti-estrogen therapy has good effect in vitro or in animal experi-ments,but hormone therapy for the patients had no better effect and longer survival time,which became a short board of the target of anti-estrogen treatment.How to judge the sex hormone dependent lung cancer and the screen for the factors affected the final treatment.Thus,the judgment and the screening condition of this cancer become the primary problem.Through the study,we found that ER beta may be an important factor in sex hormone dependent lung cancer.Meanwhile,The level of serum estrogen increase in promenopausal women and decrease of progesterone in postmenopausal women may be an important factor in the sex hormone dependent lung cancer.Si RNA was designed and synthesized as expression vector and transfected into human lung cancer LTEP-a2 cells.Compared with negative control group and blank control group,Transcription-polymerase chain reaction(RT-PCR)was used to detect ERβm RNA,Western blot was used to detect the expression of ERβ protein.Cellular growth was tested by methylthiazoltetrazolium colorimetry(MTT)assay and the cell apoptosis rates after treatment by 17β-estradiol were detected by flow cytometry.With our experiments,ERβ may be an important factor in sex hormone dependent lung cancer.Judging the female sex hormone dependent lung cancer as anti-extrogen therapy effects,we study the relationships between the tumor and the clinicopathologic characteristics to screen for factors of sex hormone dependence of female patients.Single factor analysis and the logistic regression analysis model were used to evaluate.Lung cancer is a complicated process,and it is also a new task for the sex hormone dependent female lung cancer.The study on the mechanism of sex hormones and related receptors is helpful to realize the personalized diagnosis and treatment of lung cancer.This study attempts to find the conditions of screening for such patientsfrom different aspects,and to provide a new treatment for non small cell lung cancer in women.The research includes four parts as following.Part 1.Estrogen receptor expression of female patients with non-small lung cancer and its clinical significanceObjective To evaluate estrogen receptor expression of female patients with non-small lung cancer and the relationship with clinicopathologic features and postoperative survival rate.Methods: From April 2007 to May 2012,125 female patients undergoing surgical treatment for NSCLC in Jiang Men central hospital,Sun Yat-Sen University were prospectively investigate for estrogen receptor(ERα and ERβ)and progestogen receptor(PRA and PRB)expression by using immunohistochemistry.The influence factors were analyzed.Kaplan-Meier method was used for survival analysis.Results: 1.None expression of ERα were detected in 125 female patients with NSCLC.Positive expression ERβ was present in 58 patients(46.4%),2.None expression of PRA and PRB were detected in 125 female patients with NSCLC.3.there was association between ERβ positivity with age,menopausal status,tumor stage and tumor types.No relationships were found between ERβ and tumor size,smoking situation as well as histologic differentiation.Adenocarcinomas were more likely to have ERβ positive tumors than squamous cell carcinoma and other types.there was a trend for a higher frequency with ERβ positive tumors and higher tumor staging.4.Premenopausal women with ERβ negative tumors demonstrated better survival compared with women with ERβpositive tumor,while promenopausal women with ERβ positive tumors demonstrated better survival compared with women with ERβnegative tumor(P<0.05).Conclusions: 1.The detection of estrogen receptor expression could play a significant role.It can predict the prognosis and identify who was estrogen dependent lung cancer in female patients,which may be useful in evaluating patients for postoper-ative comprehensive treatment.2.Estrogen receptor β in tumor may influence the growth and survival of female patients with non-small cell lung cancer.It may be an important factor for the screening of sex hormone dependent cancer in female patients.Part 2.Serum levels of sex hormones and expression of estrogen receptors in female patients with non-small lung cancer.Objective: There has been a marked increase in female patients with non-small lung cancer(NSCLC)and a younger trend of onset recently.Previous studies had shown that lung cancer was sexal hormone dependent tumor and affected with sex hormone.we study on the relationship between the expression of estrogen receptor and serum sexal hormone level in female patients with NSCLC and its significance.Methods: The study subjects were 94 premenopausal women and 128 postmenopausal women with NSCLC undergoing surgical treatment in Jiangmen Central Hospital,Sun Yat-Sen University.Serum levels of estradiol(E2),folliclestimulat-ing hormone(FSH),luteinizing hormone(LH)and progesterone(P)were measured by chemiluminescence,expression of ERα and ERβwas detected by immunohistochemis-try.The relationship were analyzed.Results: 1.There was no expression of ERα in all female patients with NSCLC.Positive expression ERβ was present 36 patients in pre menopausal women(38.29%)and 47 patients in promenopausal women(36.72%).2.There was no expression of PRA and PRB in all female patients with NSCLC.3.ERβ was not found in 54 cases of 79 premenopausal women with non-small cell lung in normal E2 group,while 25 cases showed grade expression.4 cases of 15 patients had no obvious expression and 11 cases showed grade expression in higeher E2 group.The expression of estrogen receptor and Serum levels of E2 between the normal group and the higher group in premenopausal women shows statistical significance(P<0.05).4.No relationships were found estrogen receptor and Serum levels of FSH,LHbetween the normal group and the higher group in premenopausal women,Serum levels of Pbetween the normal group and the lower group,respectively(p>0.05).5.ERβ was not found in 75 cases of 107 promenopausal women with non-small cell lung in normal P group,while 32 cases showed grade expression.6 cases of 21 patients had no obvious expression and 15 cases showed grade expression in lower P group.The expression of estrogen receptor and serum levels of P between the normal group and the lower group in promenopausal women shows statistical significance(P<0.05).6.No relationships were found estrogen receptor and Serum levels of E2,FSH,LH between the normal group and the higher group in promenopausal women(p>0.05).Conclusions: 1.Serum levels of sex hormones have metabolism disorders and imbalances in female patients with NSCLC.2.The disorders of serum levels of E2 in premenopausal women and P in promeno-pausal women may be factors screening for sex hormone dependence of female patients with NSCLC.3.No relationships were found estrogen receptor and serum levels of folli clestimulat-ing hormone(FSH)and luteinizing hormone(LH)between the normal group and the higher group before and after menopause in female patients with NSCLC.4.Serum levels of E2 rise in premenopausal women or P decrease in promenopausal women can affect estrogen receptor expression,which may be the factor for the screen-ing of sex hormone dependent lung cancer.Part 3.Factors screening for sex hormone dependence of female patients with non-small lung cancerObjective: Judging the female sex hormone dependent lung cancer as anti-extrogen therapy effects,we study the relationships between the tumor and the Clinicopathologic characteristics to screen for factors of sex hormone dependence of female patients.Methods: From August 2007 to August 2014,29 cases in group A of female patients with sexual hormone dependent lung cancer had relieve who underwent anti-estrogen treatment,while 83 cases in group B of female patients with sexual hormone non-dependent lung cancer had invalid or worse who had underwent anti-estrogen treatment in Jiangmen Central Hospital,Sun Yat-Sen University.Single factor analysis and the logistic regression analysis model were used to evaluate factors of sex hormone dependence of female patients.Results: there was no statistically significant difference with patient age,tumor differentiation degree,period time,smoking status,age of menarche,menstrual cycle,(P > 0.05),tumor type,marital status,number of estrogen receptor expression,menopausal status,pregnancy was statistically significant(P < 0.05).Using multiariable Logistic regression model analysis,the results shown that tumor types,marital status,expression of estrogen receptor,menopausal status,number of reproductive was statistically significant(P < 0.05).Conclusions:Tumor types especially adenocarcinoma,marital status,the expression of estrogen receptor beta,menopausal status,number of reproductive may be screening factors of female sex hormone dependent factors of lung cancer.Part 4.Effects of ERβ gene silencing by si RNA on apoptosis of human lung cancer LTEP-a2 cells overexpression of estrogen receptor βObjective: To investigate the effect of ERβ gene silencing by small interference RNA(si RNA)on apoptosis of human lung cancer LTEP-a2 cells overexpression of estrogen receptor β and on the sensitivity to 17β-estradiol.Methods: Si RNA was designed and synthesized as expression vector and transfected into human lung cancer LTEP-a2 cells.Compared with negative control group and blank control group,Transcription-polymerase chain reaction(RT-PCR)was used to detect ERβm RNA,Western blot was used to detect the expression of ERβ protein.Cellular growth was tested by methylthiazoltetrazolium colorimetry(MTT)assay and the cell apoptosis rates after treatment by 17β-estradiol were detected by flow cytom-etry.Results: 1.Expression of ERβ m RNA was decreased significantly in si RNA group compared with negative control group and blank control group(P<0.05).2.Expression of ERβ protein was decreased significantly in si RNA group compared with negative control group and blank control group(P<0.05).No relationships were found between negative control group and blank control group(P>0.05).3.IT showed that the survival rate decreased significantly using MTT colorimetric method(MTT method)in transfected group compared with negative control group and blank control group with 17-beta estradiol intervention(P<0.05),the growth of the lung cancer cell LTEP-a2 was significantly inhibited in si RNA group compared with negative control group and blank control group with 17-beta estradiol intervene-tion(P<0.05).No relationships were found between negative control group and blank control group(P>0.05).4.In the cellular apoptic rate,the transfected group was significantly higher than those negative control group and blank control group(35.68±1.58 %,3.55±0.33%,2.34±0.58%,P<0.05).No relationships were found between negative control group and blank control group(P>0.05).Conclusions: 1 ERβ gene silenced by si RNA might inhibite growth of non-small lung cancer with estrogen intervention.2.ERβ gene silenced by si RNA might promote apoptosis of non-small lung cancer with estrogen intervention.3.ERβ-si RNA gene therapy may be a new target point for sex hormone dependence of female patients with non-small lung cancer.4.ERβ could play a significant role and identifies sex hormone dependence of female patients with non-small lung cancer,which may be useful in evaluating patients for comprehensive treatment.